Treatment of EBV and KHSV infection and associated abnormal cellular proliferation

ABSTRACT

A method and composition for the treatment, prevention and/or prophylaxis of a host, and in particular, a human, infected with Epstein-Barr virus (EBV), is provided that includes administering an effective amount of a 5-substituted uracil nucleoside or its pharmaceutically acceptable salt or prodrug, optionally in a pharmaceutically acceptable diluent or excipient.

The present application is a divisional of U.S. Pat. No. 10/326,444,filed Dec. 19, 2002, incorporated by reference in its entirety, whichclaims priority to U.S. provisional application No. 60/345,130, filed onDec. 20, 2001.

FIELD OF THE INVENTION

The present invention is directed to (i) compounds, compositions andmethods for the treatment or prophylaxis of abnormal cellularproliferation in Epstein-Barr virus (EBV) or Kaposi's sarcoma-associatedherpes (“KHSV”) positive cells; (ii) treatment of EBV- orKHSV-infections; and (iii) gene therapy treatment of abnormallyproliferating cells that are EBV and KHSV negative.

BACKGROUND OF THE INVENTION

Epstein-Barr virus (EBV) was discovered in 1964 in the neoplastic Bcells of a patient with Burkitt's lymphoma. EBV thus became the firstcandidate for a human tumor virus.

Early studies indicated the viral genome was present in two endemictumors, Burkitt's lymphoma (equatorial Africa) and nasopharyngealcarcinoma (Southern China and coastal Asia) (Henle W. & Henle G. (1985)Epstein-Barr virus and human malignancies, Adv. Viral Oncol. 5, 201). Bythe late 1970s, it became evident that EBV plays a role in thedevelopment of B-cell lymphoproliferative disorders/lymphomas (BLPD) inT-cell immunocompromised patients, including solid organ and bone marrowtransplant recipients, patients infected with HIV and children withcongenital immunodeficiencies (Hanto D. W., Gajl-Peczalska K. J.,Frizzera G., Arthur D. C., Balfour H. H., Jr., McClain K. (1983)EBV-induced polyclonal and monoclonal B-cell lymphoproliferativediseases occurring after renal transplantation. Clinical, pathologic,and virologic findings and implications for therapy, Ann Surg. 198,356-69). In recent years, EBV has been identified in the hematopoietictumor cells of approximately 50% of cases of Hodgkins disease, a largenumber of T-lymphomas and rare NK/monocytoid/dendritic cellmalignancies, as well as in sporadic epithelial carcinomas (including inparticular gastric carcinoma, and possibly aggressive breast carcinomas)(Kieff E. (2001) EBV and its Replication, In: Fields Virology, Phila.,Penn: Lippincott-Raven; p. 2511-2573; Pagano J. S. (1999) EBV: the firsthuman tumor virus and its role in cancer, Proc. Assoc. Am. Physicians,111, 573-580; Bonnet M., Guinebretiere J. M., Kremmer E., Grunewald V.,Benhamou E., Contesso G. (1999) Detection of EBV in invasive breastcancers, J. Natl. Cancer Inst. 91, 1376-1381). An association withleiomyosarcomas in immunodeficient patents has also been found (RogatschH., Bonatti H., Menet A., Larcher C., Feichtinger H., Dirnhofer S.(2000) EBV-associated multicentric leiomyosarcoma in an adult patientafter heart transplantation: case report and review of the literature.Am. J. Surg. Path. 24, 614-21). Several lines of evidence indicate thatviral gene products contribute to multi-step tumorigenesis in thesediverse neoplasms.

EBV is a member of the human herpesvirus family. Infection in childhoodis usually asymptomatic; however, approximately 50% of individuals withdelayed exposure develop a self-limited lymphoproliferative syndrome,acute infectious mononucleosis. Similar to other herpesviruses, EBVpersists in a latent form for the life of the host. Serological surveysindicate that greater than 90% of the world population is infected withEBV (Henle W. & Henle G. (1979) Seroepidemiology of the virus, In: TheEpstein-Barr Virus (Epstein M. A. & Achong B. G. eds), Springer Verlag,New York, pp. 61-73). The ubiquity of infection, coupled with increasingevidence for a broad role in virus-associated malignancies (in theimmuno-compromised as well as the normal host) demonstrates a criticalneed to develop preventive and therapeutic strategies to limit EBVinfection and the effects thereof.

Several approaches to prevent and treat the manifestations ofEBV-associated malignancies are being investigated. These includegeneration of vaccines (for prevention, though one does not exist atthis time), delivery of humoral and cell-based immune therapies,chemotherapy, gene therapy (for treatment) and antiviral drug therapy,based on decreasing EBV lytic replication in the hope that this willindirectly result in a decrease in the number of EBV-infected cells ableto develop into tumors (for prophylaxis).

Treatment of EBV and EBV-Associated Diseases

Immunomodulatory agents such as α- and γ-interferons, UVIG, retinoicacid, and others, either alone or in combination have been used to treatB-cell lymphoproliferative disease (BLPD), with variable success.However, responses have not been observed in other EBV-associated tumors(Shapiro R. S., Chauvenet A., McGuire W., Pearson A., Craft A. W.,McGlave P., Filipovich A. (1988) Treatment of B-cell lymphoproliferativedisorders with interferon alfa and intravenous gamma globulin, N. Engl.J. Med. 318, 1334; Pomponi F., Cariati R., Zancai P., De Paoli P.,Rizzo, S., Tedeschi, R. M. (1996) Retinoids irreversibly inhibit invitro growth of EBV-immortalized B lymphocytes, Blood, 88, 3147-3159).

The use of complement-fixing, anti-B cell monoclonal antibodies to treatEBV-infected B-lymphomas previously achieved limited success. AB-cell-directed Mab to CD20 (Rituximab) is now commercially availableand has achieved the greatest success in treatment of BLPD/lymphoma todate. However, anaphylacetic reactions have limited therapy in somecases and not all B-cell tumors bear CD20. Moreover, Rituximab confersno specificity for the virus-infected cell, causing long-term impairmentof new humoral immune responses; Rituximab also has no efficacy for aspectrum of non-B-cell, EBV-associated diseases (Fischer A., Blanche S.,Le Bidois J., Bordigoni P., Garnier J. L., Niaudet P. (1991) Anti-B-cellmonoclonal antibodies in the treatment of severe B-celllymphoproliferative syndrome following bone marrow and organtransplantation, N. Engl. J. Med. 324, 1451-1456; Milpied N., VasseurB., Parquet N., Garnier J. L., Antoine C., Quartier P. (2000) Humanizedanti-CD20 monoclonal antibody (Rituximab) in post transplantB-lymphoproliferative disorder: a retrospective analysis on 32 patients,Ann. Oncol. 11(Suppl 1), 113-116).

Individual cell-based immune strategies are promising, but they incur arisk of graft-versus-host-disease and of transmission of pathogensduring ex vivo propagation/preparation of cells, and will be expensive(Papadopoulos E. B., Ladanyi M., Emanuel D., Mackinnon S., Boulad F.,Carabasi M. H. (1994) Infusions of donor leukocytes to treatEBV-associated lymphoproliferative disorders after allogeneic BMT, N.Engl. J. Med. 330, 1185-1191; Aguilar L. K., Rooney C. M., Heslop H. E.(1999) Lymphoproliferative disorders involving EBV after hemopoieticstem cell transplantation, Curr. Opin. Oncol. 11, 96-101).

Gene therapy strategies to introduce novel compounds that inhibit EBVoncoproteins or that inhibit cellular genes that are critical forvirus-associated oncogenesis or that introduce cytotoxic gene products(such as the HSV1-TK gene into EBV-infected tumor cells followed byganciclovir therapy) are under study. All are in early development andsuffer from standard problems of delivery to the appropriate tumor site.

In the chemotherapy field, currently there are few or no clinicallyeffective anti-EBV agents that are without undesirable side effects.Although several drug candidates have been shown to be effective againstEBV replication in cell culture, their clinical application has beenrestricted by their lack of effect on the course of latency associateddisease, because all of these antiviral agents target only EBVreplication.

The greatest challenge in EBV therapy is the latent infection. There areno drugs, licensed or even experimental, regardless of mechanism ofaction, that have shown any specific effect on latent EBV or other gammaherpesvirus infection (Lin, J. C. (1999) Antiviral therapy forEpstein-Barr virus: the challenge ahead, Recent Res. Develop.Antimicrob. Agents and Chemother. 3, 191-223; Pagano J. S. (1995)Epstein-Barr virus: therapy of active and latent infection, in AntiviralChemotherapy (eds. Jeffries & De Clercq), John Wiley & Sons, Chichester,pp. 155-195).

Several compounds have been shown to have activity against EBVreplication in culture at concentrations non-toxic to cell growth. Theseinclude acyclovir (ACV), ganciclovir (DHPG), pencyclovir, D-FMAU and itsanalogs, 5-bromovinyl dUrd, phosphonoformate and phosphorothioateoligonucleotides. See Lin et al., Antimicrob. Agents Chemo. 32:265-267(1988); Lin et al., Antimicrob. Agents Chemo., 32:1068-1072 (1988);Cheng et al., Proc. Natl., Acad. Sci. USA, 80:2767-2770 (1983); Datta etal., Proc. Natl., Acad. Sci. USA, 77:5163-5166 (1980); Datta et al.,Virol., 114:52-59 (1981); Lin et al., Antimicrob. Agents & Chemo.,31:1431-1433 (1987); Olka & Calendar, Virol. 104:219-223 (1980); Lin etal., J. Virol., 50:50-55 (1984); Yao et al., Antimicrob. Agents & Chemo.37:1420-1425 (1993) and Yao et al., Biochem. Pharm., 51:941-947 (1966).

U.S. Pat. Nos. 5,565,438, 5,567,688 and 5,587,362 (Chu et al.) disclosethe use of 2′-fluoro-5-methyl-β-L-arabinofuranolyluridine (L-FMAU) forthe treatment of hepatitis B and Epstein-Barr virus.

WO96/13512 (Genencor International, Inc. and Lipitek, Inc.) disclosesthe preparation of L-ribofuranosyl nucleosides as antitumor agents andvirucides.

Tsai et al. in Biochem. Pharmacol. 1994, 48(7), 1477-81, disclose theeffect of anti-HIV agents 2′-β-D-F-2′,3′-dideoxynucleoside analogs onthe cellular content of mitochondrial DNA and lactate production.

WO 96/40164 and WO 95/07287 (Emory University, UAB Research Foundation,and the Centre National de la Recherche Scientifique) disclose severalβ-L-2′,3′-dideoxynucleosides for the treatment of hepatitis B virus andHIV, respectively.

Novirio Pharmaceuticals, Ltd. (WO 00/09531) disclose2′-deoxy-β-L-erythropento-furanonucleosides (also referred to as β-L-dNor β-L-2′-dN), including β-L-deoxyribothymidine (β-L-dT) andβ-L-deoxyribouridine (β-L-dU).

U.S. Pat. Nos. 5,792,773, 6,022,876 and 6,274,589 (Yale University andThe University of Georgia Research Foundation, Inc.) disclose certainβ-L dioxolanyl uracil-based nucleosides for the treatment of EBV. Thecompounds preferably have a 5-halosubstituted uracil base, andreportedly exhibit unexpectedly high activity against EBV,Varicella-Zoster virus (VZV) and Kaposi's Sarcoma virus (HV-8).

Gene Therapy

In studies of gene therapy for cancer, researchers are working torecruit the immune response to fight the disease or to make the cancercells more sensitive to ablative treatment, such as chemotherapy. Someof the gene therapy techniques under study include:

-   -   Substitution of a “working” copy of a gene for an inactive or        defective gene. For example, this technique could be used to        restore the ability of a defective gene (such as mutant p53) to        suppress or block the development of cancer cells.    -   Introduction of a “survival gene,” such as the multidrug        resistance (MDR) gene into stem cells (cells in the bone marrow        that produce blood cells). The MDR gene is used to make the stem        cells more resistant to the side effects of the high doses of        anticancer drugs.    -   Injection of cancer cells with a gene that makes them more        susceptible to treatment with an anticancer drug. Scientists        hope that treatment with the drug will kill only the cells that        contain the drug-sensitive gene. This is known as suicide gene        therapy.

Suicide gene therapy is defined as the transduction of a gene thatconverts a prodrug into a toxic substance. Independently, the geneproduct and the prodrug are nontoxic. Two such systems have been widelyinvestigated: the Escherichia coli cytosine deaminase (CD) gene plus5-fluorocytosine (5-FC) and the herpes simplex virus thymidine kinasegene (HSV1-TK) plus ganciclovir (GCV).

The CD gene product converts 5-FC to the chemotherapeutic agent,5-fluorouracil (5-FU) (Huber Be Austin, E A Good, S S, et al. (1993) Invivo antitumor activity of 5-fluorocytosine on human colorectalcarcinoma cells genetically modified to express cytosine deaminase.Cancer Res. 53:4619-4626) and has been studied primarily as a treatmentfor hepatic metastases of gastrointestinal tumors, for which 5-FU iscommonly used. A significant bystander effect is active throughproduction of locally high levels of freely diffusible 5-FU (Trinh Q T,Austin E A, Murray D M, et al., Enzyme/prodrug gene therapy: Comparisonof cytosine deaminase/5-fluorcytosine versus thymidinekinase/ganciclovir enzyme/prodrug systems in a human colorectalcarcinoma cell line. Cancer Res 55:4808-4812, 1995). Systemic therapywith 5-FC results in growth suppression of CD-transduced tumors, whereaslittle growth inhibition is achieved in the same tumors with high dosesof systemic 5-FU (Huber B E, Austin E A, Good S S et al. (1993) In vivoantitumor activity of 5-fluorocytosine on human colorectal carcinomacells genetically modified to express cytosine deaminase. Cancer Res.53:4619-4626). No systemic growth suppression was seen in non-transducedtumors growing in the same animals, indicating the lack of serum 5-FUlevels sufficient for antitumor activity. Interestingly, otherinvestigators have noted that successful treatment of CD-transducedtumors with 5-FC can result in activity against challenge tumors (MullenC A, Kilstrup M, Blaese R M (1992) Transfer of the bacterial gene forcytosine deaminase to mammalian cells confers lethal sensitivity to5-fluorocytosine: a negative selection system. Proc. Natl. Acad. Sci.USA 89:33-37). Depletion of CD8⁺ T-cells or granulocytes abrogates theeffects of CD plus 5-FC (Consalvo M., Mullen C. A., Modesti A., MusianiP., Allione A., Cavallo F., Giovarelli M., Formi G. (1995)5-Fluorocytosine-induced eradication of murine adenocarcinomasengineered to express the cytosine deaminase suicide gene requires hostimmune competence and leaves an efficient memory. J. Immunol. 154,5302-5312), indicating that inadvertent stimulation of immunologicalactivity in this system may further enhance the efficacy of thisapproach.

Strategies for treating liver metastases have focused on regionaldelivery of the CD gene into areas surrounding metastases (Ohwada A,Hirschowitz E A, Crystal R G (1996) Regional delivery of an adenovirusvector containing the Escherichia coli cytosine deaminase gene toprovide local activation of 5-fluorocytosine to suppress the growth ofcolon carcinoma metastatic to liver. Hum. Gene Ther. 7:1567-1576).Further refinements for systemic gene delivery are being exploredthrough the use of tissue-specific promoters, such as carcinoembryonicantigen (CEA) ora-fetoprotein genes, for targeting gene expression toliver tumor cells after hepatic-artery infusion of the CD vector(Richards C A, Austin E A, Huber B E (1995) Transcriptional regulatorysequences of carcinoembryonic antigen: identification and use withcytosine deaminase for tumor-specific gene therapy. Hum. Gene Ther.6:881-893; Kanai F, Lan K H, Shiratori Y, et al. (1997) In vivo genetherapy for α-fetoprotein-producing hepatocellular carcinoma byadenovirus-mediated transfer of cytosine deaminase gene. Cancer Res.57:461-465).

The selective toxicity of ganciclovir (GCV) for cells expressingHSV-1-TK has been utilized similarly to promote tumor killing in a genetherapy model. In an original report (Culver K. W., Ram Z., WallbridgeS., Ishii H., Oldfield E. H., Blaese R. M. (1992) In vivo gene transferwith retroviral vector-producer cells for treatment of experimentalbrain tumors, Science, 256, 1550-1552), rapidly dividing murine gliomacells were infected in vivo with an amphotropic retrovirus vectorcontaining HSV-TK. The animals were then treated with GCV. This resultedin the death of tumor cells expressing the viral TK, but spared adjacentnormal cells that replicated too slowly for efficient retroviralinfection. Because of the so-called bystander effects, this treatment iseffective in destroying the tumor cells that contain as few as 10%TK-expressing cells. Adjacent cells that replicate rapidly also take upthe cytotoxic phosphorylated nucleosides.

HSV1-TK phosphorylates GCV to GCV-monophosphate (GCV-MP) in arate-limiting step, which can be further converted to a nucleotideanalogue that inhibits DNA synthesis via cellular enzymes (Moolten F. L.(1986) Tumor chemosensitivity conferred by inserted herpes thymidinekinase genes: paradigm for a prospective cancer control strategy. CancerRes. 46, 5276-5281). This metabolic change causes a significantby-stander effect through several mechanisms: gap junctions transportnon-diffusible phosphorylated GCV to non-transduced cells;non-transduced cells endocytose debris containing phosphorylated GCVfrom dying cells; and an induced immune response leads to tumor killing(Vile R. G., Nelson J. A., Castleden S., Chong H., Hart I. R. (1994)Systemic gene therapy of murine melanoma using tissue specificexpression of the HSVtk gene involves an immune component. Cancer Res.54, 6228-6234; Elshami A A, Saavedra A, Zhang H, et al. Gap junctionplays a role in the “bystander effect” of the herpes simplex virusthymidine kinase/ganciclovir system in vitro. Gene Ther 1996; 3:85-92;Hamel W, Magnelli L, Chiarugi V P, Israel M A. Herpes simplex virusthymidine kinase/ganciclovir-mediated apoptotic death of bystandercells. Cancer Res 1996 56:2697-2702; Mesnil M.; Piccoli C.; Tiraby G.,Willecke K.; Yamasaki H. Bystander killing of cancer cells byherpes-simplex virus thymidine kinase gene is mediated by connexins.Proc. Natl. Acad. Sci. USA 93, 1831-1835; 1996). This type of genetherapy has been explored for a variety of cancers, including localizedbrain tumors (Culver K W, Ram Z, Walbridge S, Ishii H, Oldfield E H,Balese, R M 1992. In vivo gene transfer with retroviral vector producercells for treatment of experimental brain tumors. Science 256:1550-1552;Barba D. Hardin J, Sadelain M, et al. Development of anti-tumor immunityfollowing thymidine kinase-mediated killing of experimental braintumors. Proc Natl Acad Sci USA 1994; 91, 4348-52; Chen S, Shine H D,Goodman J C, Grossman R G, Woo S L C 1994. Gene therapy for braintumors: regression of experimental gliomas by adenovirus-mediated genetransfer in vivo. Proc Natl Acad Sci USA 91: 3054-3057) andmesotheliomas (Elshami A A, Saavedra A, Zhang H, et al. Gap junctionplays a role in the “bystander effect” of the herpes simplex virusthymidine kinase/ganciclovir system in vitro. Gene Ther 1996; 3:85-92),liver metastases (Caruso M., Panis Y., Gagandeep S., Houssin D.,Salzmann J. L., Klatzmann D. Regression of established macroscopic livermetastases after in-situ transduction of a suicide gene. Proc. Natl.Acad. Sci. USA 90, 7024-7028, (1993)), and peritoneal-based metastases(Tong X W, Block A, Chen S H, Woo S L C, Kieback D G.Adenovirus-mediated thymidine kinase gene transduction in humanepithelial ovarian cancer cell lines followed by exposure toganciclovir. Anticancer Res 1996 16, 1611-1617; Yee D, McGuire S E,Brunner N, Kozelsky T W, Allred D C, Chen S H, Woo S L C.Adenovirus-mediated gene transfer of herpes simplex virus thymidinekinase in an ascites model of human breast cancer. Hum Gene Ther 1996 7,1251-1257). There have been more than 35 clinical trials using thisapproach for human cancers worldwide. Although the growth-suppressiveactivities of HSV-TK plus GCV are significant, cure rates thus far arelow, as in situ transduction (gene delivery) remains inadequate and thebystander effect is variable. Notably, both the CD and HSV-TK systems,and p53 gene therapy, additionally sensitize cancer cells to radiation,suggesting possible combination therapies to control advanced tumors(Kim J H, Kim S H, Kolozsvary A, Brown S L, Lim O B, Freytag S O.Selective enhancement of radiation response of herpes simplex virusthymidine kinase transduced 9L gliosarcoma cells in vitro and in vivo byantiviral agents. Int J Radiat Oncol Biol Phys 33: 861-868, 1995; KhilM. S.; Kim J. H., Mullein C. A., Kim S. H., Freytag S. O.Radiosensitization by 5-fluorocytosine of human colorectal carcinomacells in culture transduced with cytosine deaminase gene. ClinicalCancer Res. 2, 53-57 (1996)).

Use of HSV-TK plus GCV for the treatment of metastatic disease presentsseveral problems. Treatment of tumors with HSV-TK suppresses growth oftumors derived from challenge injections of the parental cell line,indicating the induction of systemic anti-tumor activity in some models(Barba D. Hardin J, Sadelain M et al. Development of anti-tumor immunityfollowing thymidine kinase-mediated killing of experimental braintumors. Proc Natl Acad Sci USA 1994 91, 4348-52; Vile R. G., Nelson J.A., Castleden S., Chong H., Hart I. R. Systemic gene-therapy of murinemelanoma using tissue-specific expression of the HSVtk gene involves animmune component. Cancer Res. 54, 6228-6234; 1994). Some evidence existsthat this suppression is mediated by immune cells (Vile R. G., Nelson J.A., Castleden S., Chong H., Hart I. R. Systemic gene-therapy of murinemelanoma using tissue-specific expression of the HSVtk gene involves animmune component. Cancer Res. 54, 6228-6234; 1994; Yamamoto S., SuzukiS., Hoshino A., Akimoto M., Shimada T. (1997) Herpes simplex virusthymidine kinase/ganciclovir-mediated killing of tumor cells inducestumor-specific cytotoxic T-cells in mice. Cancer Gene Ther. 4, 91-96),but the significance and generality of these observations are largelyunknown. Furthermore, systemic delivery of HSV-tk to target metastaticlesions through intravenous (Vile R. G., Nelson J. A., Castleden S.,Chong H., Hart I. R. Systemic gene-therapy of murine melanoma usingtissue-specific expression of the HSVtk gene involves an immunecomponent. Cancer Res. 54, 6228-6234; 1994) or peritoneal (Tong X W,Block A, Chen S H, Woo S L, Kieback D G. Adenovirus-mediated thymidinekinase gene transduction in human epithelial ovarian cancer cell linesfollowed by exposure to ganciclovir. Anticancer Res 1996 16: 1611-1617;Yee D, McGuire S E, Brunner N, Kozelsky T W, Allred D C, Chen S H, Woo SL. Adenovirus-mediated gene transfer of herpes simplex virus thymidinekinase in an ascites model of human breast cancer. Hum Gene Ther 1996;7, 1251-1257) routes may lead to significant liver injury (Yee D,McGuire S E, Brunner N, Kozelsky T W, Allred D C, Chen S H, Woo S L C.Adenovirus-mediated gene transfer of herpes simplex virus thymidinekinase in an ascites model of human breast cancer. Hum Gene Ther 1996;7, 1251-1257; Brand K., Arnold W., Bartels T., Lieber A., Kay M. A.,Strauss M., Dorken B. Liver-associated toxicity of the HSV-tk/GCVapproach and adenoviral vectors. Cancer Gene Therapy 4, 9-16; 1997; QianC, Idoate M, Bilbao R, Sangro B, Bruna O. Vazquez J et al. Gene transferand therapy with adenoviral vector in rats withdiethylnitrosamine-induced hepatocellular carcinoma. Hum Gene Ther 1997;8, 349-358); tissue-specific vectors may be required for safe systemicdelivery of this gene.

Epstein Barr Virus Thymidine Kinase (EBV-TK)

EBV-associated diseases are primarily manifest as virus-infected tumorsin which the viral genome is present, however, infection is latent andfew EBV genes are expressed. Increased levels of EBV lytic replicationhave been observed in the setting of acute infectious mononucleosis, ausually self-limited lymphoproliferative disorder and in oral hairyleukoplakia, a lytic disease of the oral cavity that occurs primarily inpatients with AIDS. Increased levels of EBV lytic replication have beenobserved in the blood of immunocompromised patients and correlates withthe subsequent development of lymphoproliferative disorders in thesepatients. There is no standard therapy for any of these conditions.

EBV encodes a thymidine kinase (TK), which is strictly a kinase with thecapacity to phosphorylate thymidine and a range of thymidine analogsthat is localized to the BamHI X fragment of the genome (Tung P. P. &Summers W. C. (1994) Substrate specificity of Epstein-Barr virusthymidine kinase. Antimicrob. Agents Chemother. 38, 2175-79; GustafsonE. A., Chillemi A. C., Sage D. R., Fingeroth J. D. (1998) TheEpstein-Barr virus thymidine kinase does not phosphorylate gancicloviror acyclovir and demonstrates a narrow specificity compared to herpessimplex virus type 1 thymidine kinase. Antimicrob. Agents Chemother. 42,2923-31). Although latently EBV-infected B-cells and EBV⁺ tumors do notroutinely express EBV-TK, in vitro exposure of latently infected cellsto the tumor promoter (12-O-tetradecanoylphorbol-1,3-acetate) PMA/TPA orto the polar organic compound sodium butyrate (NaB) results in modestinduction of lytic replication (1%-40% of cells depending on the line)that is accompanied by EBV-TK expression (Stinchcobe T. & Clough W.(1985) EBV induces a unique pyrimidine 2′-deoxynucleoside kinaseactivity in superinfected and virus producer B cell lines. Biochemistry,24, 2021-2033). In several EBV⁺ B-cell lines, the use of TPA and NaBtogether has been found to synergistically activate the lytic cycle(Anisimova E., Prachova K., Roubal J., Vonka V. (1984) Effects ofn-butyrate and phorbol ester (TPA) on induction of EBV antigens and celldifferentiation. Arch. Virol. 81, 223-237). Even when production ofvirus is minimal, EBV genes active during the lytic cycle are induced bydrug treatment, suggesting that drug-induced gene expression is notidentical to expression triggered in the course of a normal, productiveinfection. Although herpesvirus replication proceeds in a definedsequence, with synthesis of immediate early (IE) genes preceding early(E) and late (L) genes, artificial inducers of productive infection canalter the normal stoichiometry of viral gene expression. Induction isoften abortive with IE, and E proteins synthesized in the absence oflate proteins and virus assembly. In recent studies, NaB and argininebutyrate (ArgB) and related compounds have been administrated to healthyadults and children without major side-effects (Daniel P., Brazier M.,Cerutti I., Pieri F., Tardivel I., Desmet G. (1989) Pharmacokineticstudy of butyric acid administered in vivo as sodium and argininebutyrate salts. Clin. Chim. Acta 181, 255-263; Perrine S. P., Ginder G.D., Faller D. V., Dover G. H., Ikuta T., Witkowska H. E. (1993) Ashort-term trial of butyrate to stimulate fetal globin-gene expressionin beta-globin disorders. N. Engl. J. Med. 328, 81-86). ArgB is FDAapproved to induce fetal hemoglobin and abort hemolytic crisis inchildren with sickle cell anemia and β-thalassemia (Perrine S. P.,Ginder G. D., Faller D. V., Dover G. H., Ikuta T., Witkowska, H. E.(1993) A short-term trial of butyrate to stimulate fetal-globin-geneexpression in the beta-globin disorders. N. Engl. J. Med. 328, 81-86).In addition, protein kinase C activators pharmacologically distinct fromTPA, such as the bryostatins, which lack tumor-promoting activity, arecurrently in clinical trials for cancer patients (Prendiville J.,Crowther D., Thatcher N., Woll P. J., Fox B. W., McGown A. (1993) Aphase I study of intravenous bryostatin 1 in patients with advancedcancer. Br. J. Cancer 68, 418-424).

Related gammaherpesvirus HHV8 expresses a thymidine kinase with similarsubstrate specificity to Epstein Barr virus (Gustafson E A, Schinazi RF, Fingeroth J D (2000) Human herpesvirus 8 open reading frame 21 is athymidine and thymidylate kinase of narrow substrate specificity thatefficiently phosphorylates zidovudine but not ganciclovir. J. Virol. 74,684-692)). This virus is currently referred to in the literature asKHSV. See also Moore S M, Cannon J S, Tanhehco Y C, Hamzeh F M, AmbinderR F. (2001) Induction of Epstein-Barr virus kinases to sensitize tumorcells to nucleoside analogues. Antimicrob Agents Chemother,45(7):2082-91; Ansari A, Emery V C. (1999) The U69 gene of humanherpesvirus 6 encodes a protein kinase which can confer ganciclovirsensitivity to baculoviruses. J Virol, 73(4):3284-91; Cannon J S, HamzehF, Moore S, Nicholas J, Ambinder R F. (1999) Human herpesvirus 8-encodedthymidine kinase and phosphotransferase homologues confer sensitivity toganciclovir. J Virol, 73(6):4786-93; and Emery V C, Griffiths P D.(2000) Prediction of cytomegalovirus load and resistance patterns afterantiviral chemotherapy. Proc Natl Acad Sci 97(14): 8039-44.

Therefore, it is an object of the present invention to providecompounds, compositions and methods for the treatment or prophylaxis ofa host, particularly a human patient or other host animal, infected withEBV (or the related gammaherpesvirus KHSV).

It is another object of the present invention to provide compounds,compositions and methods for the treatment or prophylaxis of a host,particularly a human patient or other host animal, suffering from adisease associated with the lytic replication of EBV (or the relatedgammaherpesvirus KHSV).

It is further another object of the present invention to providecompounds, compositions and methods for the treatment or prophylaxis ofa host, particularly a human patient or other host animal, sufferingfrom a disease associated with abnormally proliferating cells that areinfected with EBV (or the related gammaherpesvirus KHSV).

It is another objective of the present invention to provide cell linesthat can be used in gene therapy, in particular, cell lines transfectedwith EBV-TK, or the related gammaherpesvirus thymidine kinase (KHSV-TK).

It is a further objective of the present invention to provide compounds,compositions and methods for gene therapy of a host, particularly ahuman patient or other host animal exhibiting a cancer or infectionrelated to Epstein Barr Virus.

Finally, it is an objective of the present invention to provide kits andassays to assess the effects of compounds and/or compositions inreducing abnormal cellular proliferation using transduced cell lines.

SUMMARY OF THE INVENTION

It has been found that certain 5-substituted uracil-nucleosides areselectively phosphorylated by EBV thymidine kinase and/or KHSV thymidinekinase, and are not substantially phosphorylated by human cellularthymidine kinase. Therefore, based on this and other observations,several embodiments of the invention are provided.

(i) A method for the treatment of a host with an abnormal cellularproliferation or abnormal cell disorder, wherein the cell is EBV or KHSVpositive, that includes administering an effective amount of one or acombination of the described 5-substituted uracil-nucleosides or itspharmaceutically acceptable salt or prodrug, optionally in apharmaceutically acceptable carrier. In this embodiment, 5-substituteduracil-nucleosides that are substrates for the host cellular polymeraseare selected. When the uracil nucleoside is administered to the cell, itis selectively phosphorylated by the viral thymidine kinase and then isincorporated into cellular DNA by the action of the cellular polymerase,which reduces or terminates the proliferation. This can initiate thecellular repair mechanism, resulting in apoptosis. In an alternative oradditional embodiment, the uracil nucleoside may act as an inhibitor ofthymidylate synthetase. This method can be used to treat any abnormalcellular proliferation or disorder, including tumor or cancer growth, inwhich the abnormal cell includes the EBV genome. Examples of diseasesthat can be treated Burkitt's lymphoma, nasopharyngeal carcinoma, anyB-cell lymphoproliferative disorders/lymphoma (BLPD), including those inT-cell immunocompromised patients, transplant recipients, patientsinfected with HIV and children with congenital immunodeficiencies,EBV-induced polyclonal and monoclonal B-cell lymphoproliferativediseases occurring after transplantation (including renal), Hodgkinsdisease, T-lymphomas and rare NK/monocytoid/dendritic cell malignancies,sporadic epithelial carcinomas, leiomyosarcomas in immunodeficientpatients and multicentric leiomyosarcoma in immunocompromised patients.Abnormal cells bearing the KHSV genome include Kaposi's infected cells,primary effusion lymphoma (which usually have both KHSV and EBV),multicentric Castleman's Disease (plasmablastic variant alwaysKHSV-infected) and germanotrophic lymphoma.

(ii) A method, use, compound and composition for the treatment of a hostwith an EBV or KHSV infection, comprising administering an effectiveamount of one or a combination of the described 5-substituteduracil-nucleosides or its pharmaceutically acceptable salt or prodrug,optionally in a pharmaceutically acceptable carrier. In this embodiment,the uracil nucleoside is administered to the cell, selectivelyphosphorylated by the viral thymidine kinase and then acts as aninhibitor of one or more viral enzymes, for example, EBV or KHSVpolymerase. In this embodiment, the 5-substituted uracil-nucleoside actsas a competitive inhibitor of viral growth. Examples of disorders thatcan be treated with this embodiment include mononucleosis (a diseasecaused by EBV that creates an excess of large lymphocytes that have aresemblance to monocytes in circulating blood), including complicatedacute infectious mononucleosis, oral hairy leukoplakia, for reduction ofEBV lytic replication following iatrogenic immunosuppression-acircumstance that may predispose a host to the development ofEBV-associated lymphomas, and any other manifestation of viralinfection.

(iii) A method use, compound and composition for the treatment of a hostwith an abnormal cellular proliferation or abnormal cell disorder,wherein the cell is EBV or KHSV negative. In this embodiment, theEBV-thymidine kinase or KHSV thymidine kinase gene is administered to ordirectly surrounding the abnormal cell in a manner that causes theincorporation of the gene into the natural cellular nucleic acid. Theviral thymidine kinase is then expressed in the host cell, and acts tophosphorylate the separately administered 5-substituteduracil-nucleoside. The therapy then proceeds as described in (i) above.

It is of importance to note that over half of diagnosed Hodgkins diseaseinvolves EBV and that substantial lymphoproliferative disease isassociated with HIV infected patients. Certain of the uracil nucleosidesdescribed herein also have activity against HIV, and thus can be used totreat patients that exhibit both HIV infection and a resultinglymphoproliferative disease.

In one embodiment of the invention, the selected compound is of theformula (I), (II), or (III):

or its pharmaceutically acceptable salt or prodrug thereof, wherein

-   i) X is O, S, NR⁴, CH₂, CHF or CF₂;-   ii) R¹ is H, halogen (Cl, Br, I, F), alkyl (including C₁, C₂, C₃,    C₄, C₅, and C₆), haloalkyl (including C₁, C₂, C₃, C₄, C₅, and C₆),    alkenyl (including C₂, C₃, C₄, C₅, and C₆), haloalkenyl (including    C₂, C₃, C₄, C₅, and C₆), alkynyl (including C₂, C₃, C₄, C₅, and C₆),    haloalkynyl (including C₂, C₃, C₄, C₅, and C₆), cycloalkyl    (including C₃, C₄, C₅, C₆, C₇, and C₈), CN, CF₃, N₃, NO₂, aryl    (including C₆, C₇, C₈, C₉, and C₁₀), heteroaryl (including C₄, C₅,    C₆, C₇, C₈, C₉, and C₁₀), acyl (including C₂, C₃, C₄, C₅, and C₆),    and COR₅ where R₅ is chosen from one of H, OH, SH, alkyl (including    C₁, C₂, C₃, C₄, C₅, and C₆), aminoalkyl (including C₁, C₂, C₃, C₄,    C₅, and C₆), alkoxy (including C₁, C₂, C₃, C₄, C₅, and C₆), or    thioalkyl (including C₁, C₂, C₃, C₄, C₅, and C₆);-   iii) R² and R^(2′) are independently H, carbonyl substituted with an    alkyl (including C₁, C₂, C₃, C₄, C₅, and C₆), alkenyl (including C₂,    C₃, C₄, C₅, and C₆), alkynyl (including C₂, C₃, C₄, C₅, and C₆),    aryl (including C₆, C₇, C₈, C₉, and C₁₀); benzyl, wherein the phenyl    group is optionally substituted with one or more substitutents;    phosphate (including monophosphate, diphosphate, triphosphate or a    stabilized phosphate prodrug); phosphate ester; sulfonate ester    including alkyl or arylalkyl sulfonyl including methanesulfonyl; a    lipid including a phospholipid; amino acid; peptide; cholesterol, or    other pharmaceutically acceptable leaving group, preferably such    that when administered in vivo, is capable of providing a compound    wherein R² and/or R^(2′) is independently H;-   iv) R³ is OH, halo (F, Cl, Br, I), a protected hydroxyl group or    CH₂OR⁴;-   v) each R⁴ is independently H, acyl (including C₂, C₃, C₄, C₅, and    C₆), alkyl (including C₁, C₂, C₃, C₄, C₅, and C₆), alkenyl    (including C₂, C₃, C₄, C₅, and C₆), alkynyl (including C₂, C₃, C₄,    C₅, and C₆), or cycloalkyl (including C₃, C₄, C₅, C₆, C₇, and C₈);-   vi) Y is H, OH, halogen (F, Cl, Br, I), N₃, CN, or OR^(2′); and-   vii) Z is O, S, alkyl (including C₁, C₂, C₃, C₄, C₅, and C₆),    alkenyl (C₂, C₃, C₄, C₅, and C₆), alkynyl (including C₂, C₃, C₄, C₅,    and C₆), CH₂, CHF, or CF₂.

The compound of the present invention can be in the form of the β-L orβ-D configuration, or any mixture of the two configurations, including aracemic mixture.

R² and R^(2′) are independently hydrogen or pharmaceutically acceptableleaving group, which when administered in vivo, is capable of providinga compound wherein R² and/or R^(2′) is independently H. For example, R²and R^(2′) independently can be hydrogen, acyl, including a carbonylsubstituted with a alkyl (including C₁, C₂, C₃, C₄, C₅, and C₆), alkenyl(including C₂, C₃, C₄, C₅, and C₆), alkynyl (including C₂, C₃, C₄, C₅,and C₆), aryl (including C₆, C₇, C₈, C₉, and C₁₀); benzyl, wherein thephenyl group is optionally substituted with one or more substitutentswhich may be cleaved by cellular esterase. Alternatively, R² and R^(2′)independently can be hydrogen or an acid or base labile leaving group,though preferably an acid-labile leaving group, such as phosphate(including monophosphate, diphosphate, triphosphate or a stabilizedphosphate prodrug); phosphate ester; sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl; a lipid including aphospholipid; amino acid; peptide or cholesterol. In a preferredembodiment, R² and/or R^(2′) are independently hydrogen or a group thatincreases the activity, bioavailability and/or stability of the selectedcompound, which when administered in vivo, is capable of providing acompound wherein R² and/or R^(2′) is independently H.

The selected uracil nucleoside derivatives are produced by syntheticmethods that are readily known to those of ordinary skill in the art, anumber of which are described below.

In one preferred embodiment, β-D-2′-deoxy-5-vinyluridine (also referredto as 5-vinyl-dU) is administered. This compound shows selectivity forEBV-TK, i.e. is specifically phosphorylated and converted into acytotoxic agent by EBV-TK. Another specific example of a selectedcompound includes β-D-2′-deoxy-5-ethynyluridine (5-ethynyl-dU) that isalso active against human immunodeficiency virus type 1 with an EC₅₀ of0.61 μM.

In other preferred embodiments, the selected compounds areβ-L-2′-deoxy-5-vinyluridine, β-L-5-vinyluridine,β-L-2′-deoxy-5-iodouridine, β-D-2′-deoxy-5-(hydroxymethyl)uridine,β-D-5-butyl-2′-deoxyuridine, β-D-5-E-(2-carboxyvinyl)-2′-deoxyuridine,(2S,4S)-5-(2-furanyl)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]uracil,(2S,4S)-5-(5-bromo-2-furanyl)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]uracil,(2S,4S)-5-(2-thienyl)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]uracil,(2S,4S)-5-(5-bromthien-2-yl)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]uracil,and(2S,4S)-5-(3-hydroxypropenyl)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]uracil.These compounds show selectivity for EBV-TK (i.e., are specificallyphosphorylated and converted into a cytotoxic agent by EBV-TK).

In another embodiment, the present therapy is used in combination oralternation with other known or developed therapies for EBV or KHSVinfected cells, including but not limited to Ganciclovir with or withoutarginine butyrate, Valaciclovir (Valtrex), donor white blood cells thathave been treated in the laboratory to kill cells infected with EBV,white blood cells from donors who are immune to Epstein-Barr virus,LFMAU, and the compounds in U.S. Pat. Nos. 5,792,773, 6,022,876 and6,274,589 (Yale University and The University of Georgia ResearchFoundation, Inc.).

Gene Therapy

The invention includes suicide gene therapy that employs the EBV-TK orKHSV-TK gene plus a selected uracil nucleoside derivative as describedherein. The EBV-TK or KHSV-TK gene is delivered into areas within andsurrounding the abnormal cellular proliferation or disorder to betreated, is taken up by the cells and can be incorporated into thecellular nucleic acid. The virus then expresses the viral thymidinekinase which phosphorylates the separately administered uracilnucleoside. A bystander effect can be achieved through production oflocally high levels of the resulting phosphorylated uracil nucleoside.Because of the bystander effect, this treatment is effective indestroying the tumor cells that contain as few as 10% TK-expressingcells. Adjacent cells that replicate rapidly take up the cytotoxicphosphorylated nucleoside. The EBV-TK and KHSV-TK genes additionallysensitize abnormal cells to radiation, suggesting possible combinationtherapies to control advanced tumors.

Thus, similar to GCV-HSV-1-TK gene therapy, gene therapy using theselected nucleoside analogues and EBV-TK may be employed in thetreatment of EBV, EBV-associated disease and cancer cells transfectedwith the gene encoding EBV-TK. Upon phosphorylation of the nucleosideanalogue by these EBV-TK-expressing cells, the nucleoside triphosphateexerts its toxicity, resulting in the death of the transfected cancer ortumor cells.

The amount of EBV-TK or KHSV-TK can be increased in latently infectedcells or transfected cells by administration of a compound that causesincreased expression of the enzyme. Examples are the tumor promoter(12-O-tetradecanoylphorbol-13-acetate) PMA/TPA and the polar organiccompound sodium butyrate (NaB). In another embodiment, arginine butyrate(ArgB) is administered. Other inducing agents include, but are notlimited to, azacytidine, hydroxyurea, interferons, gamma radiation,retinoic acid and related retinoids.

In an alternative embodiment, cell lines transfected with EBV-TK, andtheir use in gene therapy are also provided. For example, a cell can beselected that will optimally target to the location of interest in thehost for efficient therapy. The cell therapy can be autologous orheterologous. In autologous therapy, the host's own cells are removed,transfected with the EBV-TK or KHSV-TK gene as described in detail belowor as otherwise known in the art, and then transferred back into thehost. During heterologous therapy, third party donor cells aretransfected with the gene of interest and then transplanted into thehost in need of therapy.

The cells transfected with the EBV-TK or HH8V-TK gene express the enzymeafter transplantation into the host. The viral thymidine kinase acts asa selective phosphorylating agent for a uracil nucleoside administeredbefore, during and/or after cell transplantation, which in finaltriphosphorylated form, inhibits EBV or KHSV growth. This produces atherapeutic effect on any cells that include and express the EBV genome,regardless of the type of cell infection. The EBV-TK transfected celllines can be used in screening and cytotoxicity assays. Cellstransfected with EBV-TK or KHSV-TK that do not have an adversebiological effect on the host animal can be used in gene therapy inaccordance with the present invention.

As one non-limiting example, the Epstein-Barr virus (EBV) thymidinekinase (TK) gene from the viral strain B-958 was cloned into the vectorpCMV as described (Gustafson E A, Chillemi A C, Sage D R, Fingeroth J D.The Epstein-Barr virus thymidine kinase does not phosphorylateganciclovir or acyclovir and demonstrates a narrow substrate specificitycompared to the herpes simplex virus type 1 thymidine kinase. AntimicrobAgents Chemother 1998; 42(11), 2923-31). The vector was then transfectedusing Lipofectamine reagent into 2 human cell lines. The cells wereselected and expression of the TK gene (RNA and protein) was determined.The cells were used to create two assay systems to assess the ability ofEBV-TK to sensitize cells to candidate nucleoside analogs. Cells thatexpressed KHSV-TK were similarly prepared for assay.

As an additional non-limiting example, immortalized human embryonickidney cells (293 cells) were used to produce 293 EBV-TK cells and 293KHSV-TK cells. The 293 cells were co-transfected with pCMV EBV-TK andpSV2 neo and the 293 KHSV-TK cells were prepared similarly using thevector pCMV KHSV-TK together with pSV2 neo (co-transfection) or by usingthe single vector pcDNA3-KHSV-TK (which contains an endogenousselectable neo marker). The cells were selected and clones weredocumented to express KHSV-TK by RNA blot hybridization.

Positive EBV-TK clones were pooled to produce bulk populations and wereused in cytotoxicity assays to screen candidate nucleoside analogs. Twocell systems are employed in the cytotoxicity assays to determine thetoxicity of each nucleoside. The information obtained depends upon thesystem used. In 143B T-cells, cytotoxic drugs that are dependent uponphosphorylation by a TK are easily identified. 143B T-cells expressingeither EBV-TK or hTK1 permit assessment of whether either enzymeindependently may cause a drug to become cytotoxic. Thus, drugs that canbe activated by hTK1, which would cause non-specific cytotoxicity, arereadily identified, but these cells are grown in HAT media that “forces”nucleosides through the TK salvage pathway (EBV-TK or hTK1) and presentsthe possibility of a false positive. The 293 cells have endogenous hTK1and EBV-TK is maintained by selection in G418. Thus, the normal hTK1salvage pathway is operational, and nucleosides can be expected to fluxthrough the cells normally. Drugs are incubated with 293 cellstransformed with a neomycin (Neo) expressing plasmid (Neo control) andwith cells transfected with the same plasmid also expressing EBV-TK. Anucleoside selectively activated by EBV-TK should cause toxicity on 293EBV-TK and not on 293 Neo control cells. In both the 143B and 293 cellsystems, toxicity is evaluated by cell survival.

An example of an assay in which the transfected cell lines may beemployed is the colony formation assay, done with 293 transfectants.This assay is designed to identify nucleosides whose toxicity is due toincorporation into cellular DNA forming a lethal lesion as described forGCV (Rubsam L Z, Davidson B L, and Shewach D S. “Superior cytotoxicitywith ganciclovir compared with acyclovir and1-β-D-arabinofuranosylthymine in herpes simplex virus-thymidinekinase-expressing cells: a novel paradigm for cell killing” Cancer Res1998; 58(17):3873-82). Such toxicity is not dependent upon constantexposure to drug, but only exposure to drug during log phase growth.Nucleosides that act via a different mechanism, such as chaintermination or inhibition of an enzyme in a metabolic pathway, are oftenonly cytostatic, and the cells may return to normal growth after drugremoval. This assay exposes cells in log growth and then fixes them. Anucleoside that works effectively causes a decrease in the number ofcolonies, which is a direct indication of the number of cells able tosurvive and replicate after the initial exposure to drug.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a non-limiting example of a synthesis of selected compounds ofthe present invention, and in particular, a synthesis of5-E-(2-chlorovinyl)uracil nucleosides.

FIG. 2 is a non-limiting example of a synthesis of selected compounds ofthe present invention, and in particular, a synthesis of 5-vinyluracilnucleosides.

FIG. 3 is a non-limiting example of a synthesis of selected compounds ofthe present invention, and in particular, a synthesis of 5-ethynyluracilnucleosides.

FIG. 4 is a non-limiting example of a synthesis of selected compounds ofthe present invention, and in particular, a synthesis of acyclic5-substituted uracil nucleosides.

FIG. 5 is a non-limiting example of a synthesis of selected compounds ofthe present invention, and in particular, a synthesis of 5-substituteduracil 4′-thionucleosides.

FIG. 6 is a non-limiting example of a synthesis of selected compounds ofthe present invention, and in particular, a synthesis of 5-substituteduracil carbocyclic nucleosides.

FIG. 7 is a non-limiting example of a synthesis of selected compounds ofthe present invention, and in particular, a synthesis of 5-substituteddioxolane nucleosides.

FIG. 8 is a non-limiting example of a synthesis of selected compounds ofthe present invention, and in particular, a synthesis of5-substituted-thienyl uracil nucleosides.

FIG. 9 is a non-limiting example of a synthesis of selected compounds ofthe present invention, and in particular, a synthesis of 5-thienylsubstituted dioxolane nucleosides.

FIG. 10 is a non-limiting example of a synthesis of selected compoundsof the present invention, and in particular, a synthesis of5-substituted 2′-deoxyuridine nucleosides.

FIG. 11 is a graphical representation of the competition effect ofselected compounds with purified GST EBV-TK fusion proteins.

FIG. 12 is a graphical representation of the competition effect ofselected compounds, comparing 143B T-minus cells expressing EBV-TK with143B T-minus cells expressing re-introduced human TK-1 (hTK-1).

FIG. 13 is a graphical representation of the effects of5-vinyl-2′-deoxyuridine (also referred to as 5-vinyl-dU) on colonyformation in cells expressing EBV-TK versus endogenous hTK-1.

FIG. 14 is a graphical representation of the cytotoxicity of selectedcompounds on 143B cells expressing EBV-TK, hTK-1, or TK deficient (TK-)at 20 μM concentration.

FIG. 15 is a direct demonstration of the cytotoxic effect of selectedcompounds on 293 EBV-TK cells versus 293 Neo cells.

FIG. 16 is a representation of the dose-dependent inhibition of5-vinyl-2′-deoxyuridine (5-vinyl-dU) on cells transfected with EBV-TK(293 EBV-TK cells) compared with 293 Neo cells.

FIG. 17 is a graphical representation of the cytotoxicity of selectedcompounds on 143B versus 143 EBV-TK cells after 5 days treatment at 20μM.

FIG. 18 is a graphical representation of a competition assay betweenselected compounds and radio-labeled thymidine with purified GST-EBV-TKfusion protein

DETAILED DESCRIPTION OF THE INVENTION

It has been found that certain 5-substituted uracil-nucleosides areselectively phosphorylated by EBV thymidine kinase and/or KRSV thymidinekinase, and are not substantially phosphorylated by human cellularthymidine kinase. EBV-TK is strictly a thymidine kinase with thecapacity to phosphorylate thymidine and some thymidine analogs. Similarto the combination of HSV1-TK with ganciclovir (GCV), certain5-substituted uracil nucleosides effectively kill EBV-TK-expressingcells, but not cells that do not express this enzyme. In one embodimentof the present invention, the nucleoside analogs are conditionally toxicto cells, dependent upon phosphorylation selectively by EBV-TK and thecompounds' specificity for a cellular polymerase or transcriptase. Suchtoxicity will result in the selective destruction of tumor cells thatexpress EBV-TK, but not human TK1 or -2 or other human nucleoside ornucleotide kinases. Notably, this is a distinct paradigm from that oftraditional antiviral nucleoside analogues that seek to selectivelyinhibit the virus replication cycle and not interfere with that of thecell.

Several embodiments of the invention are provided.

(i) A method for the treatment of a host with an abnormal cellularproliferation or abnormal cell disorder, wherein the cell is EBV or KHSVpositive, that includes administering an effective amount of one or acombination of the described 5-substituted uracil-nucleosides or itspharmaceutically acceptable salt or prodrug, optionally in apharmaceutically acceptable carrier.

(ii) A method, use, compound and composition for the treatment of a hostwith an EBV or KHSV infection, comprising administering an effectiveamount of one or a combination of the described 5-substituteduracil-nucleosides or its pharmaceutically acceptable salt or prodrug,optionally in a pharmaceutically acceptable carrier. In this embodiment,the uracil nucleoside is administered to the cell, selectivelyphosphorylated by the viral thymidine kinase and then acts as aninhibitor of one or more viral enzymes, for example, EBV or KHSVpolymerase. In this embodiment, the 5-substituted uracil-nucleoside actsas a competitive inhibitor of viral growth.

(iii) A method, use, compound and composition for the treatment of ahost with an abnormal cellular proliferation or abnormal cell disorder,wherein the cell is EBV or KHSV negative. In this embodiment, theEBV-thymidine kinase or KHSV thymidine kinase gene is administered to ordirectly surrounding the abnormal cell in a manner that causes theincorporation of the gene into the natural cellular nucleic acid. Theviral thymidine kinase is then expressed in the host cell, and acts tophosphorylate the separately administered 5-substituteduracil-nucleoside. The therapy then proceeds as described in (i) above.

In one embodiment of the present invention, the tumor cells alreadyexpress EBV-TK and therefore there is no need for gene delivery, unlikein the HSV1-TK and GCV system.

The present invention therefore provides compounds, pharmaceuticalcompositions and methods for the treatment, inhibition or prophylaxis ofa disease associated with infection or lytic replication of anEpstein-Barr virus (or the related gammaherpesvirus KHSV) includingdiseases associated with abnormally proliferating cells infected withEBV, in a host, particularly a human patient or other host animal,comprising administering an effective amount of at least one compound asdescribed in the present invention, or its pharmaceutically acceptablesalt or prodrug thereof, optionally in a pharmaceutically acceptablecarrier or excipient.

In another aspect of the present invention, there is provided apharmaceutical composition for the treatment, inhibition and/orprophylaxis of a disease associated with infection or lytic replicationof an Epstein-Barr virus (or the related gammaherpesvirus KHSV)including diseases associated with abnormally proliferating cellsinfected with EBV in a host, particularly a human patient or other hostanimal, comprising administering an effective amount of at least onecompound of the present invention, or its pharmaceutically acceptablesalt or prodrug thereof, in combination with one or more other effectiveanti-EBV agent and/or agent that can induce and/or upregulate EBV-TKexpression, or its pharmaceutically acceptable salt or prodrug thereof,optionally in a pharmaceutically acceptable carrier or excipient.

In still another aspect of the present invention, there is provided amethod for the treatment, inhibition and/or prophylaxis of a diseaseassociated with infection or lytic replication of an Epstein-Barr virus(or the related gammaherpesvirus KHSV) including diseases associatedwith abnormally proliferating cells infected with EBV in a host,particularly a human patient or other host animal, comprisingadministering an effective amount of at least one compound of theinvention, its pharmaceutically acceptable salt or prodrug thereof,optionally in combination or alternation with one or more othereffective anti-EBV agent and/or agent that can induce and/or upregulateEBV-TK expression, or its pharmaceutically acceptable salt or prodrugthereof, optionally in a pharmaceutically acceptable carrier orexcipient.

In yet another aspect of the present invention, there is provided a useof at least one of the compounds of the present invention, or itspharmaceutically acceptable salt or prodrug, for the treatment,inhibition and/or prophylaxis of an EBV infection in a host,particularly a human patient or other host animal, optionally incombination or alternation with one or more other effective anti-EBVagent and/or agent that can induce and/or upregulate EBV-TK expression,or its pharmaceutically acceptable salt or prodrug thereof, optionallyin a pharmaceutically acceptable carrier or excipient.

In yet another aspect of the present invention, there is provided a useof at least one of the compounds, or its pharmaceutically acceptablesalts or prodrugs, for the treatment, inhibition and/or prophylaxis of adisease associated with infection or lytic replication of anEpstein-Barr virus (or the related gammaherpesvirus KHSV) includingdiseases associated with abnormally proliferating cells infected withEBV in a host, particularly a human patient or other host animal,optionally in combination or alternation with one or more othereffective anti-EBV agent and/or agent that can induce and/or upregulateEBV-TK expression, or its pharmaceutically acceptable salt or prodrugthereof, optionally in a pharmaceutically acceptable carrier orexcipient.

In yet another aspect of the present invention, there is provided a useof at least one of the compounds of the present invention, or itspharmaceutically acceptable salt or prodrug, in the manufacture of amedicament for the treatment, inhibition and/or prophylaxis of a diseaseassociated with infection or lytic replication of an Epstein-Barr virus(or the related gammaherpesvirus KHSV) including diseases associatedwith abnormally proliferating cells that bear the EBV genome in a host,particularly a human patient or other host animal, optionally incombination or alternation with one or more other effective anti-EBVagent and/or agent that can induce and/or upregulate EBV-TK expression,or its pharmaceutically acceptable salt or prodrug thereof, optionallyin a pharmaceutically acceptable carrier or excipient.

In an additional embodiment of the present invention, cell linestransfected with EBV-TK are provided. Another embodiment of the presentinvention, there is provided a use of a cell line transfected withEBV-TK for the treatment, inhibition and/or prophylaxis of a diseaseassociated with infection or lytic replication of an Epstein-Barr virus(or the related gammaherpesvirus KHSV) including diseases associatedwith abnormally proliferating cells infected with EBV in a host,particularly a human patient or other host animal, is provided, byadministering to said cell line an effective amount of an agentselectively activated by EBV-TK into a cytotoxic agent, or itspharmaceutically acceptable salt or prodrug thereof, optionally incombination or alternation with one or more other effective agent, orits pharmaceutically acceptable salt or prodrug thereof, optionally in apharmaceutically acceptable carrier or excipient. In a preferredembodiment of the present invention, the agent selectively activated byEBV-TK is a compound of the present invention. In one embodiment of theinvention, the effective agent administered in combination oralternation with the agent selectively activated by EBV-TK is aneffective anti-proliferative agent. In an additional embodiment of theinvention, the effective agent administered in combination oralternation with the agent selectively activated by EBV-TK is aneffective anti-EBV agent, and in particular an agent selectivelyactivated by EBV-TK. In yet another embodiment of the invention, theeffective agent administered in combination or alternation with theagent selectively activated by EBV-TK is an effective agent that caninduce and/or upregulate EBV-TK expression.

Therefore, assays and kits for detecting inhibition of abnormal cellularproliferation using the cell lines transfected with EBV-TK are alsoprovided. In another embodiment of the present invention, assays andkits are provided to analyze and predict the desired cytotoxic andvirotoxic activities in cells in which EBV-TK is expressed eitherindependently of or together with viral and cellular enzymes that arepotentially able to utilize and modify nucleoside analog substrates,such as cellular kinases.

A method for the treatment, inhibition and/or prophylaxis of a diseaseassociated with infection or lytic replication of an Epstein-Barr virus(or the related gammaherpesvirus KHSV) including diseases associatedwith abnormally proliferating cells infected with EBV in a host,particularly a human patient or other host animal, is providedcomprising transfecting cells with EBV-TK, if necessary, andadministering an effective amount of an agent selectively activated byEBV-TK into a cytotoxic agent, or its pharmaceutically acceptable saltor prodrug thereof, optionally in combination or alternation with one ormore other effective agent, or its pharmaceutically acceptable salt orprodrug thereof, optionally in a pharmaceutically acceptable carrier orexcipient. In a preferred embodiment of the present invention, the agentselectively activated by EBV-TK is a compound of the present invention.In one embodiment of the invention, the effective agent administered incombination or alternation with the agent selectively activated byEBV-TK is an effective anti-proliferative agent. In an additionalembodiment of the invention, the effective agent administered incombination or alternation with the agent selectively activated byEBV-TK is an effective anti-EBV agent, and in particular an agentselectively activated by EBV-TK. In yet another embodiment of theinvention, the effective agent administered in combination oralternation with the agent selectively activated by EBV-TK is aneffective agent that can induce and/or upregulate EBV-TK expression.

In yet another embodiment of the invention, there is provided a use ofat least one of the compounds of the present invention, or itspharmaceutically acceptable salt or prodrug thereof, for the treatment,inhibition and/or prophylaxis of a disease associated with infection orlytic replication of an Epstein-Barr virus (or the relatedgammaherpesvirus KHSV) including diseases associated with abnormallyproliferating cells infected with EBV in a host, particularly a humanpatient or other host animal, is provided, optionally in combination oralternation with one or more other effective agent, or itspharmaceutically acceptable salt or prodrug thereof, optionally in apharmaceutically acceptable carrier or excipient. In a preferredembodiment of the present invention, the agent selectively activated byEBV-TK is a compound of the present invention. In one embodiment of theinvention, the effective agent administered in combination oralternation with the agent selectively activated by EBV-TK is aneffective anti-proliferative agent. In an additional embodiment of theinvention, the effective agent administered in combination oralternation with the agent selectively activated by EBV-TK is aneffective anti-EBV agent, and in particular an agent selectivelyactivated by EBV-TK. In yet another embodiment of the invention, theeffective agent administered in combination or alternation with theagent selectively activated by EBV-TK is an effective agent that caninduce and/or upregulate EBV-TK expression.

In yet another embodiment of the invention, there is provided a use ofat least one of the compounds of the present invention, or itspharmaceutically acceptable salt or prodrug, in the manufacture of amedicament for the treatment, inhibition and/or prophylaxis of a diseaseassociated with infection or lytic replication of an Epstein-Barr virus(or the related gammaherpesvirus KHSV) including diseases associatedwith abnormally proliferating cells infected with EBV, where cells canbe transfected with EBV-TK, if necessary, in a host, particularly ahuman patient or other host animal, is provided, optionally incombination or alternation with one or more other effective agent, orits pharmaceutically acceptable salt or prodrug thereof, optionally in apharmaceutically acceptable carrier or excipient. In a preferredembodiment of the present invention, the agent selectively activated byEBV-TK is a compound of the present invention. In one embodiment of theinvention, the effective agent administered in combination oralternation with the agent selectively activated by EBV-TK is aneffective anti-proliferative agent. In an additional embodiment of theinvention, the effective agent administered in combination oralternation with the agent selectively activated by EBV-TK is aneffective anti-EBV agent, and in particular an agent selectivelyactivated by EBV-TK. In yet another embodiment of the invention, theeffective agent administered in combination or alternation with theagent selectively activated by EBV-TK is an effective agent that caninduce and/or upregulate EBV-TK expression.

In another embodiment of the present invention, a use of a cell linetransfected with EBV-TK in the manufacture of a medicament for thetreatment, inhibition and/or prophylaxis of a disease associated withinfection or lytic replication of an Epstein-Barr virus (or the relatedgammaherpesvirus KHSV) including diseases associated with abnormallyproliferating cells infected with EBV in a host, particularly a humanpatient or other host animal, is provided, by administering to said cellline an effective amount of an agent selectively activated by EBV-TKinto a cytotoxic agent, or its pharmaceutically acceptable salt orprodrug thereof, optionally in combination or alternation with one ormore other effective agent, or its pharmaceutically acceptable salt orprodrug thereof, optionally in a pharmaceutically acceptable carrier orexcipient. In a preferred embodiment of the present invention, the agentselectively activated by EBV-TK is a compound of the present invention.In one embodiment of the invention, the effective agent administered incombination or alternation with the agent selectively activated byEBV-TK is an effective anti-proliferative agent. In an additionalembodiment of the invention, the effective agent administered incombination or alternation with the agent selectively activated byEBV-TK is an effective anti-EBV agent, and in particular an agentselectively activated by EBV-TK. In yet another embodiment of theinvention, the effective agent administered in combination oralternation with the agent selectively activated by EBV-TK is aneffective agent that can induce and/or upregulate EBV-TK expression.

I Selected Compounds

In one embodiment of the invention, the selected compound is of formula(I), (II), or (III):

or its pharmaceutically acceptable salt or prodrug thereof, wherein

-   i) X is O, S, NR⁴, CH₂, CHF or CF₂;-   ii) R¹ is H, halogen (Cl, Br, I, F), alkyl (including C₁, C₂, C₃,    C₄, C₅, and C₆), haloalkyl (including C₁, C₂, C₃, C₄, C₅, and C₆),    alkenyl (including C₂, C₃, C₄, C₅, and C₆), haloalkenyl (including    C₂, C₃, C₄, C₅, and C₆), alkynyl (including C₂, C₃, C₄, C₅, and C₆),    haloalkynyl (including C₂, C₃, C₄, C₅, and C₆), cycloalkyl    (including C₃, C₄, C₅, C₆, C₇, and C₈), CN, CF₃, N₃, NO₂, aryl    (including C₆, C₇, C₈, C₉, and C₁₀), heteroaryl (including C₄, C₅,    C₆, C₇, C₈, C₉, and C₁₀), acyl (including C₂, C₃, C₄, C₅, and C₆),    and COR⁵ where R⁵ is chosen from one of H, OH, SH, alkyl (including    C₁, C₂, C₃, C₄, C₅, and C₆), aminoalkyl (including C₁, C₂, C₃, C₄,    C₅, and C₆), alkoxy (including C₁, C₂, C₃, C₄, C₅, and C₆), or    thioalkyl (including C₁, C₂, C₃, C₄, C₅, and C₆);-   iii) R² and R^(2′) are independently H, carbonyl substituted with an    alkyl (including C₁, C₂, C₃, C₄, C₅, and C₆), alkenyl (including C₂,    C₃, C₄, C₅, and C₆), alkynyl (C₂, C₃, C₄, C₅, and C₆), aryl    (including C₆, C₇, C₈, C₉, and C₁₀); benzyl, wherein the phenyl    group is optionally substituted with one or more substitutents;    phosphate (including monophosphate, diphosphate, triphosphate or a    stabilized phosphate prodrug); phosphate ester; sulfonate ester    including alkyl or arylalkyl sulfonyl including methanesulfonyl; a    lipid including a phospholipid; amino acid; peptide; cholesterol, or    other pharmaceutically acceptable leaving group, preferably such    that when administered in vivo, is capable of providing a compound    wherein R² and R^(2′) is independently H;-   iv) R³ is OH, halo (F, Cl, Br, I), protected hydroxyl group or    CH₂OR⁴;-   v) each R⁴ is independently H, acyl (including C₂, C₃, C₄, C₅, and    C₆), alkyl (including C₁, C₂, C₃, C₄, C₅, and C₆), alkenyl    (including C₂, C₃, C₄, C₅, and C₆), alkynyl (including C₂, C₃, C₄,    C₅, and C₆), or cycloalkyl (C₃, C₄, C₅, C₆, C₇, and C₈);-   vi) Y is H, OH, halogen (F, Cl, Br, I), N₃, CN, or OR^(2′); and-   vii) Z is O, S, NO₂, alkyl (including C₁, C₂, C₃, C₄, C₅, and C₆),    alkenyl (including C₂, C₃, C₄, C₅, and C₆), alkynyl (C₂, C₃, C₄, C₅,    and C₆), CH₂, CHF, or CF₂.

The compound of the present invention can be in the form of the β-L orβ-D configuration, or a racemic mixture.

In one embodiment of the invention, it is desired for the compound to beselectively activated by EBV-TK (or the related KHSV-TK); therefore R²and R^(2′) are independently hydrogen or pharmaceutically acceptableleaving group, which when administered in vivo, is capable of providinga compound wherein R² and/or R^(2′) is independently H. For example, R²and R^(2′) independently can be hydrogen or carbonyl substituted with aalkyl (including C₁, C₂, C₃, C₄, C₅, and C₆), alkenyl (including C₂, C₃,C₄, C₅, and C₆), alkynyl (including C₂, C₃, C₄, C₅, and C₆), aryl(including C₆, C₇, C₈, C₉, and C₁₀); benzyl, wherein the phenyl group isoptionally substituted with one or more substitutents which may becleaved by cellular esterase. Alternatively, R² and R^(2′) independentlycan be hydrogen or an acid or base labile leaving group, thoughpreferably an acid-labile leaving group, such as phosphate (includingmonophosphate, diphosphate, triphosphate or a stabilized phosphateprodrug); phosphate ester; sulfonate ester including alkyl or arylalkylsulfonyl including methanesulfonyl; a lipid including a phospholipid;amino acid; peptide or cholesterol. In a preferable embodiment, R²and/or R^(2′) are independently hydrogen or a group that increases theactivity, bioavailability and/or stability of the selected compound,which when administered in vivo, is capable of providing a compoundwherein R² and/or R^(2′) is independently H.

In one preferred embodiment, the selected compound isβ-D-2′-deoxy-5-vinyluridine (also referred to as 5-vinyl-dU) of thestructure:

or a pharmaceutically acceptable salt or prodrug thereof.

In an additional embodiment, the selected compound isβ-D-2′-deoxy-5-ethynyluridine (also referred to as 5-ethynyl-dU) of thestructure:

or a pharmaceutically acceptable salt or prodrug thereof. This compoundshows also activity against human immunodeficiency virus with an EC₅₀ of0.61 μM.

In one preferred embodiment, the selected compound isβ-L-2′-deoxy-5-vinyluridine of the structure:

or a pharmaceutically acceptable salt or prodrug thereof.

In another preferred embodiment, the selected compound isβ-L-5-vinyluridine of the structure:

or a pharmaceutically acceptable salt or prodrug thereof.

In another preferred embodiment, the selected compound isβ-L-2′-deoxy-5-iodouridine of the structure:

or a pharmaceutically acceptable salt or prodrug thereof.

In another preferred embodiment, the selected compound isβ-D-2′-deoxy-5-(hydroxymethyl)uridine of the structure:

or a pharmaceutically acceptable salt or prodrug thereof.

In one preferred embodiment, the selected compound isβ-D-5-butyl-2′-deoxyuridine of the structure:

or a pharmaceutically acceptable salt or prodrug thereof.

In one preferred embodiment, the selected compound isβ-D-5-E-(2-carboxyvinyl)-2′-deoxyuridine of the structure:

or a pharmaceutically acceptable salt or prodrug thereof.

In one preferred embodiment, the selected compound is(2S,4S)-5-(2-furanyl)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]uracil ofthe structure:

or a pharmaceutically acceptable salt or prodrug thereof.

In one preferred embodiment, the selected compound is(2S,4S)-5-(5-bromo-2-furanyl)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]uracilof the structure:

or a pharmaceutically acceptable salt or prodrug thereof.

In one preferred embodiment, the selected compound is(2S,4S)-5-(2-thienyl)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]uracil ofthe structure:

or a pharmaceutically acceptable salt or prodrug thereof.

In one preferred embodiment, the selected compound is,(2S,4S)-5-(5-bromthien-2-yl)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]uracilof the structure:

or a pharmaceutically acceptable salt or prodrug thereof.

In one preferred embodiment, the selected compound is(2S,4S)-5-(3-hydroxypropenyl)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]uracilof the structure:

or a pharmaceutically acceptable salt or prodrug thereof.II Definitions

The term “independently” is used herein to indicate that the variable,which is independently applied, varies independently from application toapplication. Thus, in a compound such as R″XYR″, wherein R″ is“independently carbon or nitrogen,” both R″ can be carbon, both R″ canbe nitrogen, or one R″ can be carbon and the other R″ nitrogen.

As used herein, the term “enantiomerically pure” refers to a nucleosidecomposition that comprises at least approximately 95%, and preferablyapproximately 97%, 98%, 99% or 100% of a single enantiomer of thatnucleoside.

As used herein, the term “substantially free of” or “substantially inthe absence of” refers to a nucleoside composition that includes atleast 85 or 90% by weight, preferably 95% to 98% by weight, and evenmore preferably 99% to 100% by weight, of the designated enantiomer ofthat nucleoside. In a preferred embodiment, in the methods and compoundsof this invention, the compounds are substantially free of enantiomers.

Similarly, the term “isolated” refers to a nucleoside composition thatincludes at least 85 or 90% by weight, preferably 95% to 98% by weight,and even more preferably 99% to 100% by weight, of the nucleoside, theremainder comprising other chemical species or enantiomers.

The term “alkyl,” as used herein, unless otherwise specified, refers toa saturated straight, branched, or cyclic, primary, secondary, ortertiary hydrocarbon of typically C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉,and C₁₀, and specifically includes methyl, trifluoromethyl, ethyl,propyl, isopropyl, cyclopropyl, butyl, isobutyl, t-butyl, pentyl,cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl,cyclohexylmethyl, 3-methylpentyl, 2,2-dimethylbutyl, and2,3-dimethylbutyl. The term includes both substituted and unsubstitutedalkyl groups. Moieties with which the alkyl group can be substituted areselected from the group consisting of hydroxyl, amino, alkylamino,arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate,phosphonic acid, phosphate, or phosphonate, either unprotected, orprotected as necessary, as known to those skilled in the art, forexample, as taught in Greene et al., Protective Groups in OrganicSynthesis, John Wiley & Sons, 2^(nd) Edition, 1991, hereby incorporatedby reference.

The term “lower alkyl,” as used herein, and unless otherwise specified,refers to a C₁, C₂, C₃, and C₄ saturated straight, branched, or ifappropriate, a cyclic (for example, cyclopropyl) alkyl group, includingboth substituted and unsubstituted forms. Unless otherwise specificallystated in this application, when alkyl is a suitable moiety, lower alkylis preferred. Similarly, when alkyl or lower alkyl is a suitable moiety,unsubstituted alkyl or lower alkyl is preferred.

The terms “alkylamino” or “arylamino” refer to an amino group that hasone or two alkyl or aryl substitutents, respectively.

The term “alkoxy” as used herein includes linear or branchedoxy-containing radicals having alkyl moieties, such as methoxy radical.The term “alkoxyalkyl” also embraces alkyl radicals having one or morealkoxy radicals attached to the alkyl radical, that is, to formmonoalkoxyalkyl and dialkoxyalkyl radicals. The “alkoxy” radicals may befurther substituted with one or more halo atoms, such as fluoro, chloroor bromo, to provide “haloalkoxy” radicals. Examples of such radicalsinclude fluoromethoxy, chloromethoxy, trifluoromethoxy, difluoromethoxy,trifluoroethoxy, fluoroethoxy, tetrafluoroethoxy, pentafluoroethoxy, andfluoropropoxy.

The term “protected,” as used herein and unless otherwise defined,refers to a group that is added to an oxygen, nitrogen, or phosphorusatom to prevent its further reaction or for other purposes. A widevariety of oxygen and nitrogen protecting groups are known to thoseskilled in the art of organic synthesis.

The term “aryl,” as used herein, and unless otherwise specified, refersto phenyl, biphenyl, or naphthyl, and preferably phenyl. The termincludes both substituted and unsubstituted moieties. The aryl group canbe substituted with one or more moieties selected from the groupconsisting of hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy,nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, orphosphonate, either unprotected, or protected as necessary, as known tothose skilled in the art, for example, as taught in Greene, et al.,Protective Groups in Organic Synthesis, John Wiley and Sons, SecondEdition, 1991.

The terms “alkaryl” or “alkylaryl” refer to an alkyl group with an arylsubstitutent. The terms “aralkyl” or “arylalkyl” refer to an aryl groupwith an alkyl substitutent.

The term “halo,” as used herein, includes chloro, bromo, iodo andfluoro.

The term “acyl” refers to a carboxylic acid ester in which thenon-carbonyl moiety of the ester group is selected from straight,branched, or cyclic alkyl or lower alkyl, alkoxyalkyl includingmethoxymethyl, aralkyl including benzyl, aryloxyalkyl such asphenoxymethyl, aryl including phenyl optionally substituted with halogen(F, Cl, Br, I), alkyl (including C₁, C₂, C₃, and C₄) or alkoxy(including C₁, C₂, C₃, and C₄), sulfonate esters such as alkyl oraralkyl sulfonyl including methanesulfonyl, the mono, di or triphosphateester, trityl or monomethoxytrityl, substituted benzyl, trialkylsilyl(e.g. dimethyl-t-butylsilyl) or diphenylmethylsilyl. Aryl groups in theesters optimally comprise a phenyl group.

The terms “heteroaryl” or “heteroaromatic,” as used herein, refer to anaromatic that includes at least one sulfur, oxygen, nitrogen orphosphorus in the aromatic ring.

The term “lower acyl” refers to an acyl group in which the non-carbonylmoiety is lower alkyl.

The term “host,” as used herein, refers to a unicellular ormulticellular organism in which the virus can replicate, including celllines and animals, and preferably a human. Alternatively, the host canbe carrying a part of the viral genome, whose replication or functioncan be altered by the compounds of the present invention. The term hostspecifically refers to infected cells, cells transfected with all orpart of the viral genome and animals, in particular, primates andhumans. In most animal applications of the present invention, the hostis a human-patient. Veterinary applications, in certain indications,however, are clearly anticipated by the present invention.

III Pharmaceutically Acceptable Salts or Prodrugs

The term “pharmaceutically acceptable salt or prodrug” is usedthroughout the specification to describe any pharmaceutically acceptableform (such as an ester, phosphate ester, salt of an ester or a relatedgroup) of a compound which, upon administration to a patient, providesthe active compound. Pharmaceutically acceptable salts include thosederived from pharmaceutically acceptable inorganic or organic bases andacids. Pharmaceutically acceptable prodrugs refer to a compound that ismetabolized, for example hydrolyzed or oxidized, in the host to form thecompound of the present invention. Typical examples of prodrugs includecompounds that have biologically labile protecting groups on afunctional moiety of the selected compound. Prodrugs include compoundsthat can be oxidized, reduced, aminated, deaminated, hydroxylated,dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated,acylated, deacylated, phosphorylated, dephosphorylated to produce theactive compound. The compounds of this invention possess antiviralactivity against Epstein-Barr virus and/or cytotoxic activity againstcells expressing EBV-TK, i.e., EBV-infected cells, and, in particular,EBV-infected cells that abnormally proliferate (develop into tumors), orEBV-TK transduced cells, or are metabolized to a compound that exhibitssuch activity.

In cases where compounds are sufficiently basic or acidic to form stablenontoxic acid or base salts, administration of the compound as apharmaceutically acceptable salt may be appropriate. Examples ofpharmaceutically acceptable salts are organic acid addition salts formedwith acids, which form a physiological acceptable anion, for example,tosylate, methanesulfonate, acetate, citrate, malonate, tartarate,succinate, benzoate, ascorbate, α-ketoglutarate and α-glycerophosphate.Suitable inorganic salts may also be formed, including, sulfate,nitrate, bicarbonate and carbonate salts.

Alternatively, pharmaceutically acceptable salts may be obtained, forexample by reacting a sufficiently basic compound such as an amine witha suitable acid affording a physiologically acceptable anion. Alkalimetal (for example, sodium, potassium or lithium) or alkaline earthmetal (for example calcium and magnesium) salts of, for example,carboxylic acids can also be made.

Any of the compounds described herein can be administered as a prodrugto increase the activity, bioavailability, stability or otherwise alterthe properties of the selected compound. A number of prodrug ligands areknown. In general, alkylation, acylation or other lipophilicmodification of the mono, di or triphosphate of the compound, forexample the nucleoside will increase the stability of the compound, forexample as a nucleotide. Examples of substitutent groups that canreplace one or more hydrogens on the phosphate moiety are alkyl, aryl,steroids, carbohydrates, including sugars, 1,2-diacylglycerol andalcohols. Many are described in R. Jones & N. Bischofberger, AntiviralResearch, 27 (1995) 1-17. Any of these can be used in combination withthe disclosed nucleosides to achieve a desired effect.

The selected compounds can also be provided as a 5′-phosphoether lipidor a 5′-ether lipid, as disclosed in the following references, which areincorporated by reference herein: Kucera L. S., N. Iyer, E. Leake, A.Raben, Modest E. K., Daniel L. W., C. Piantadosi, “Novelmembrane-interactive ether lipid analogs that inhibit infectious HIV-1production and induce defective virus formation,” AIDS Res. Hum.Retroviruses, 1990, 6, 491-501; Piantadosi C., J. Marasco C. J., S. L.Morris-Natschke, K. L. Meyer, F. Gumus, J. R. Surles, K. S. Ishaq, L. S.Kucera, N. Iyer, C. A. Wallen, S. Piantadosi, E. J. Modest, “Synthesisand evaluation of novel ether lipid nucleoside conjugates for anti-HIVactivity,” J. Med. Chem., 1991, 34, 1408-1414; Hostetler K. Y., D. D.Richman, D. A. Carson, L. M. Stuhmiller, G. M. T. van Wijk, H. van denBosch, “Greatly enhanced inhibition of human immunodeficiency virus type1 replication in CEM and HT4-6C cells by 3′-deoxythymidine diphosphatedimyristoylglycerol, a lipid prodrug of 3′-deoxythymidine,” Antimicrob.Agents Chemother., 1992, 36, 2025-2029; Hostetler K. Y., L. M.Stuhrmiller, H. B. Lenting, H. van den Bosch, D. D. Richman, “Synthesisand antiretroviral activity of phospholipid analogs of azidothymidineand other antiviral nucleosides.” J. Biol. Chem., 1990, 265, 61127.

Nonlimiting examples of U.S. patents that disclose suitable lipophilicsubstitutents that can be covalently incorporated into the compound,preferably at the 5′-OH position of the compound or lipophilicpreparations, include U.S. Pat. Nos. 5,149,794 (Yatvin et al.);5,194,654 (Hostetler et al., 5,223,263 (Hostetler et al.); 5,256,641(Yatvin et al.); 5,411,947 (Hostetler et al.); 5,463,092 (Hostetler etal.); 5,543,389 (Yatvin et al.); 5,543,390 (Yatvin et al.); 5,543,391(Yatvin et al.); and 5,554,728 (Basava et al.), all of which areincorporated by reference. Foreign patent applications that discloselipophilic substitutents that can be attached to the nucleosides of thepresent invention, or lipophilic preparations, include WO 89/02733, WO90/00555, WO 91/16920, WO 91/18914, WO 93/00910, WO 94/26273, WO96/15132, EP 0 350 287, EP 93917054.4, and WO 91/19721, all of which areincorporated by reference.

Prodrugs also include amino acid esters of the disclosed nucleosides(see, e.g., European Patent Specification No. 99493, the text of whichis incorporated by reference, which describes amino acid esters ofacyclovir, specifically the glycine and alanine esters which showimproved water-solubility compared with acyclovir itself, and U.S. Pat.No. 4,957,924 (Beauchamp), which discloses the valine ester ofacyclovir, characterized by side-chain branching adjacent to thealpha-carbon atom, which showed improved bioavailability after oraladministration compared with the alanine and glycine esters). A processfor preparing such amino acid esters is disclosed in U.S. Pat. No.4,957,924 (Beauchamp), the text of which is incorporated by reference.As an alternative to the use of valine itself, a functional equivalentof the amino acid may be used (e.g., an acid halide such as the acidchloride, or an acid anhydride). In such a case, to avoid undesirableside-reactions, it may be is advantageous to use an amino-protectedderivative.

IV Combination or Alternation Therapy

In another embodiment, for the treatment, inhibition, prevention and/orprophylaxis of Epstein-Barr viral infection, the selected compound orits derivative or salt can be administered in combination or alternationwith another antiviral agent, such as anti-EBV agent, including those ofthe formula above. In general, in combination therapy, effective dosagesof two or more agents are administered together, whereas duringalternation therapy, an effective dosage of each agent is administeredserially. The dosage will depend on absorption, inactivation andexcretion rates of the drug as well as other factors known to those ofskill in the art. It is to be noted that dosage values will also varywith the severity of the condition to be alleviated. It is to be furtherunderstood that for any particular subject, specific dosage regimens andschedules should be adjusted over time according to the individual needand the professional judgment of the person administering or supervisingthe administration of the compositions.

Nonlimiting examples of antiviral agents that can be used in combinationwith the compounds disclosed herein include acyclovir (ACV), AZT, 3TC,d4T, ganciclovir (GCV or DHPG) and its prodrugs (e.g.,valyl-ganciclovir), E-5-(2-bromovinyl)-2′-deoxyuridine (BVDU),(E)-5-vinyl-1-β-D-arabonosyluracil (VaraU),(E)-5-(2-bromovinyl)-1-β-D-arabinosyluracil (BV-araU),1-(2-deoxy-2-fluoro-β-D-arabinosyl)-5-iodocytosine (D-FIAC),1-(2-deoxy-2-fluoro-β-L-arabinosyl)-5-methyluracil (L-FMAU),(S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA],(S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-2,6-diaminopurine[(S)-HPMPDAP], (S)-1-(3-hydroxy-2-phosphonyl-methoxypropyl)cytosine[(S)-HPMPC, or cidofivir], and(2S,4S)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]-5-iodouracil(L-5-IoddU).

In one embodiment, the compounds of the invention may be employedtogether with at least one other antiviral agent chosen from polymeraseinhibitors.

In addition, compounds according to the present invention can beadministered in combination or alternation with one or moreanti-retrovirus, anti-hepatitis B virus (HBV), anti-hepatitis C virus(HCV), anti-EBV or anti-herpetic agent or interferon, anti-cancer orantibacterial agents, including other compounds of the presentinvention. Certain compounds according to the present invention may beeffective for enhancing the biological activity of certain agentsaccording to the present invention by reducing or otherwise altering themetabolism, catabolism or inactivation of other compounds and as such,are co-administered for this intended effect.

Additionally, an anti-proliferative agent can be administered incombination or alternation therapy for the present invention. Theanti-proliferative agent, as used herein, is any agent that decreasesthe hyperproliferation of cells. Any of the anti-proliferative agentslisted below, or any other such agent known or discovered to exhibit ananti-proliferative effect can be used in accordance with this invention.

Proliferative disorders are currently treated by a variety of classes ofcompounds including alkylating agents, antimetabolites, naturalproducts, enzymes, biological response modifiers, miscellaneous agents,hormones and antagonists, such as those listed below. Examples, include,but are not limited to

Monoclonal Antibodies

Monoclonal antibodies directed to proliferating cells such as Rituximab(anti-CD20) for B-cell tumors.

Alkylating Agents

Nitrogen mustards: mechlorethamine (Hodgkins disease, non-Hodgkinslymphomas), Cyclophosphamide Ifosfamide (acute and chronic lymphocyticleukemias, Hodgkins disease, non-Hodgkins lymphomas, multiple myeloma,neuroblastoma, breast, ovary, lung, Wilms' tumor, cervix, testis,soft-tissue sarcomas), melphalan (L-sarcolysin) (multiple myeloma,breast, ovary), chlorambucil (chronic lymphocytic leukemia, primarymacroglobulinemia, Hodgkins disease, non-Hodgkins lymphomas).

Ethylenimines and methylmelamines: hexamethylmelamine (ovary), Thiotepa(bladder, breast, ovary).

Alkyl sulfonates: busulfan (chronic granuloytic leukemia).

Nitrosoureas: Carmustine (BCNU) (Hodgkin's disease, non-Hodgkinslymphomas, primary brain tumors, multiple myeloma, malignant melanoma),Lomustine (CCNU) (Hodgkins disease, non-Hodgkins lymphomas, primarybrain tumors, small-cell lung), Semustine (methyl-CCNU) (primary braintumors, stomach, colon), Streptozocin (streptozocin) (malignantpancreatic insulinoma, malignant carcinoin).

Triazenes: Dacarbazine (DTIC; dimethyltriazenoimid-azolecarboxamide)(malignant melanoma, Hodgkins disease, soft-tissue sarcomas).

Anti-Metabolites

Folic acid analogs: methotrexate (amethopterin) (acute lymphocyticleukemia, choriocarcinoma, mycosis fungoides, breast, head and neck,lung, osteogenic sarcoma). Pyrimidine analogs: fluorouracil(5-fluorouracil; 5-FU) Floxuridine (fluorodeoxyuridine; FUdR) (breast,colon, stomach, pancreas, ovary, head and neck, urinary bladder,premalignant skin lesions) (topical), Cytarabine (cytosine arabinoside)(acute granulocytic and acute lymphocytic leukemias).

Purine analogs and related inhibitors: mercaptopurine (6-mercaptopurine;6-MP) (acute lymphocytic, acute granulocytic and chronic granulocyticleukemia), thioguanine (6-thioguanine: TG) (acute granulocytic, acutelymphocytic and chronic granulocytic leukemia), Pentostatin(2′-deoxycyoformycin) (hairy cell leukemia, mycosis fungoides, chroniclymphocytic leukemia).

Vinca alkaloids: Vinblastine (VLB) (Hodgkins disease, non-Hodgkinslymphomas, breast, testis), vincristine (acute lymphocytic leukemia,neuroblastoma, Wilms' tumor, rhabdomyosarcoma, Hodgkins disease,non-Hodgkins lymphomas, small-cell lung).

Epipodophyl-lotoxins: etoposide (testis, small-cell lung and other lung,breast, Hodgkins disease, non-Hodgkins lymphomas, acute granulocyticleukemia, Kaposi's sarcoma), teniposide (testis, small-cell lung andother lung, breast, Hodgkins disease, non-Hodgkins lymphomas, acutegranulocytic leukemia, Kaposi's sarcoma).

Natural Products

Antibiotics: dactinomycin (actinonmycin D) (choriocarcinoma, Wilms'tumor rhabdomyosarcoma, testis, Kaposi's sarcoma), daunorubicin(daunomycin; rubidomycin) (acute granulocytic and acute lymphocyticleukemias), doxorubicin (soft tissue, osteogenic, and other sarcomas;Hodgkins disease, non-Hodgkins lymphomas, acute leukemias, breast,genitourinary thyroid, lung, stomach, neuroblastoma), bleomycin (testis,head and neck, skin and esophagus lung, and genitourinary tract,Hodgkins disease, non-Hodgkins lymphomas), plicamycin (mithramycin)(testis, malignant hypercalcema), mitomycin (mitomycin C) (stomach,cervix, colon, breast, pancreas, bladder, head and neck).

Enzymes: L-asparaginase (acute lymphocytic leukemia).

Biological response modifiers: interferon-alfa (hairy cell leukemia,Kaposi's sarcoma, melanoma, carcinoid, renal cell, ovary, bladder, nonHodgkins lymphomas, mycosis fungoides, multiple myeloma, chronicgranulocytic leukemia), interferon-gamma, IL-2 and IL-12.

Hormones and Antagonists

Estrogens: Diethylstibestrol ethinyl estradiol (breast, prostate)

Antiestrogen: Tamoxifen (breast).

Androgens: Testosterone propionate Fluxomyesterone (breast).

Antiandrogen: Flutamide (prostate).

Gonadotropin-Releasing Hormone Analog: Leuprolide (prostate).

Miscellaneous Agents

Platinum coordination complexes: cisplatin (cis-DDP) carboplatin(testis, ovary, bladder, head and neck, lung, thyroid, cervix,endometrium, neuroblastoma, osteogenic sarcoma).

Anthracenedione: mixtozantrone (acute granulocytic leukemia, breast).

Substituted Urea: hydroxyurea (chronic granulocytic leukemia,polycythemia vera, essential thrombocytosis, malignant melanoma).

Methylhydrazine derivative: procarbazine (N-methylhydrazine, MIH)(Hodgkins disease).

Adrenocortical suppressant: miotane (o,p′-DDD) (adrenal cortex),aminoglutethimide (breast).

Adrenorticosteriods: prednisone (acute and chronic lymphocyticleukemias, non-Hodgkins lymphomas, Hodgkins disease, breast).

Progestins: hydroxprogesterone caproate, medroxyprogersterone acetate,megestrol acetate (endometrium, breast).

Demethylating agents: azacytidine

PKC activators: bryostatins

Differentiating agents: butyrates, retinoic acid and related retinoids

Microtubule inhibitors: taxols and taxanes

Topoisomerase inhibitors: topotecan

Miscellaneous: valproic Acid, HMBA, NF-kappaB Inhibitors.

Radiation Therapy

Both the CD and HSV-TK systems additionally sensitize cancer cells toradiation, providing possible combination therapies to control advancedtumors (Kim J H, Kim S H, Kolozsvary A, Brown S L, Lim O B, Freytag S O.Selective enhancement of radiation response of herpes simplex virusthymidine kinase transduced 9L gliosarcoma cells in vitro and in vivo byantiviral agents. Int J Radiat Oncol Biol Phys 33: 861-868, 1995; KhilM. S.; Kim J. H.; Mullein C. A.; Kim S. H.; Freytag S. O.Radiosensitization by 5-fluorocytosine of human colorectal carcinomacells in culture transduced with cytosine deaminase gene. ClinicalCancer Res. 2, 53-57; 1996). Therefore, in one embodiment of theinvention, the compounds are administered in combination or alternationwith radiation therapy, i.e., as used herein, radiation is included as aviable effective antiproliferative agent.

V Pharmaceutical Compositions

Host, including humans, infected with an Epstein-Barr virus, or a genefragment thereof, can be treated by administering to the patient aneffective amount of the selected compound or a pharmaceuticallyacceptable prodrug or salt thereof in the presence of a pharmaceuticallyacceptable carrier or diluent. The active materials can be administeredby any appropriate route, for example, orally, parenterally,intravenously, intradermally, subcutaneously, or topically, in liquid orsolid form.

A preferred dose of the compound will be in the range from about 1 to 50mg/kg, preferably 1 to 20 mg/kg, of body weight per day, more generally0.1 to about 100 mg per kilogram body weight of the recipient per day.The effective dosage range of the pharmaceutically acceptable salts andprodrugs can be calculated based on the weight of the parent nucleosideto be delivered. If the salt or prodrug exhibits activity in itself, theeffective dosage can be estimated as above using the weight of the saltor prodrug, or by other means known to those skilled in the art.

The compound is conveniently administered in unit any suitable dosageform, including but not limited to one containing 7 to 3000 mg,preferably 70 to 1400 mg of active ingredient per unit dosage form. Anoral dosage of 50-1000 mg is usually convenient.

Ideally the active ingredient should be administered to achieve peakplasma concentrations of the selected compound of from about 0.2 to 70M, preferably about 1.0 to 10 M. This may be achieved, for example, bythe intravenous injection of a 0.1 to 5% solution of the activeingredient, optionally in saline, or administered as a bolus of theactive ingredient.

The concentration of selected compound in the drug composition willdepend on absorption, inactivation and excretion rates of the drug aswell as other factors known to those of skill in the art. It is to benoted that dosage values will also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed composition. The active ingredient may be administered atonce, or may be divided into a number of smaller doses to beadministered at varying intervals of time.

A preferred mode of administration of the selected compound is oral.Oral compositions will generally include an inert diluent or an ediblecarrier. They may be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, theselected compound can be incorporated with excipients and used in theform of tablets, troches or capsules. Pharmaceutically compatiblebinding agents, and/or adjuvant materials can be included as part of thecomposition.

The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a disintegrating agent such asalginic acid, Primogel or corn starch; a lubricant such as magnesiumstearate or Sterotes; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring. When the dosageunit form is a capsule, it can contain, in addition to material of theabove type, a liquid carrier such as a fatty oil. In addition, dosageunit forms can contain various other materials which modify the physicalform of the dosage unit, for example, coatings of sugar, shellac, orother enteric agents.

The compound can be administered as a component of an elixir,suspension, syrup, wafer, chewing gum or the like. A syrup may contain,in addition to the selected compounds, sucrose as a sweetening agent andcertain preservatives, dyes and colorings and flavors.

The compound or a pharmaceutically acceptable prodrug or salts thereofcan also be mixed with other active materials that do not impair thedesired action, or with materials that supplement the desired action,such as antibiotics, anti-fungals, anti-inflammatories or otherantivirals, including other nucleoside compounds. Solutions orsuspensions used for parenteral, intradermal, subcutaneous, or topicalapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetra acetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The parental preparation can be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic.

If administered intravenously, preferred carriers are physiologicalsaline or phosphate buffered saline (PBS).

In a preferred embodiment, the selected compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters and polylacetic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially.

Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) are also preferred aspharmaceutically acceptable carriers. These may be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811 (incorporated by reference). For example,liposome formulations may be prepared by dissolving appropriate lipid(s)(such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidylcholine, arachadoyl phosphatidyl choline, and cholesterol) in aninorganic solvent that is then evaporated, leaving behind a thin film ofdried lipid on the surface of the container. An aqueous solution of theselected compound or its monophosphate, diphosphate, and/or triphosphatederivatives is then introduced into the container. The container is thenswirled by hand to free lipid material from the sides of the containerand to disperse lipid aggregates, thereby forming the liposomalsuspension.

VI Processes for the Preparation of Selected Compounds

The compounds according to the present invention are produced bysynthetic methods that are readily known to those of ordinary skill inthe art and include various chemical synthetic methods.

A method for the facile preparation of 5-substituted uracil nucleosidesis also provided.

The 5-substituted uracil nucleosides disclosed herein can be prepared asdescribed in detail below, or by other assays known to those skilled inthe art. For example, such methods are described in the followingreferences: Bergstrom D. E., Ruth J. L. (1981) U.S. Pat. No. 4,247,544;De Clercq E. D. A., Verhelst G. A., Jones A. S., Walker R. T. (1984)U.S. Pat. No. 4,382,925; Jones A. S., Walker R. T., De Clercq E. D. A.,Barr P. J. (1984) U.S. Pat. No. 4,424,211; Sakata S., Machida H. (1985)U.S. Pat. No. 4,542,210; Walker R. T., Coe P. L. (1994) U.S. Pat. No.5,356,882; Spector T., Porter D. J. T., Rahim S. G. (2001) U.S. Pat. No.6,177,436; Robins M. J., Barr P. J. (1983) J. Org. Chem. 48, 1854-1862;Ogilvie K. K., Hamilton R. G., Gillen M. F., Radatus B. K. (1984) Can.J. Chem. 62, 16-21; Watanabe K. A., Su T.-L., Reichman U., Greenberg N.,Lopez C., Fox J. J. (1984) J. Med. Chem. 27, 91-94; McGee D. P. C.,Martin J. C., Smee D. F., Matthews T. R., Verheyden P. H. (1985) J. Med.Chem. 28, 1242-1245; Beauchamp L. M., Serling B. L., Kelsey J. E., BironK. K., Collins P., Selway J., Lin J.-C., Schaeffer H. J. (1988) J. Med.Chem. 31, 144-149; Balzarini J., Baumgartner H., Bodenteich M., DeClercq E., Griengl H. (1989) J. Med. Chem. 32, 1861-1865; Dyson M. R.,Coe P. L., Walker R. T. (1991) J. Med. Chem. 34, 2782-2786; Lin T.-S.,Luo M.-Z., Liu M.-C. (1995) Tetrahedron, 51, 1055-1068; Rahim S. G.,Trivedi N., Bogunovic-Batchelor M. V., Hardy G. W., Mills G., Selway J.W. T., Snowden W., Littler E., Coe P. L., Basnak I., Whale R. F., WalkerR. T. (1996) J. Med. Chem. 39, 789-795; Ma T., Pai S. B., Zhu Y. L., LinJ. S., Shanmuganathan K., Du J., Wang C., Kim H., Newton M. G., Cheng Y.C., Chu C. K. (1996) J. Med. Chem. 39, 2835-2843; Basnak I., Otter G.P., Duncombe R. J., Westwood N. B., Pietrarelli M., Hardy G. W., MillsG., Rahim S. G., Walke R. T. (1998) Nucleosides and Nucleotides, 17,29-38; Choi Y., Li L., Grill S., Gullen E., Lee C. S., Gumina G., TsujiiE., Cheng Y.-C., Chu C. K. (2000) J. Med. Chem. 43, 2538-2546.

VII Processes for the Preparation of Transfected Cell Lines with EBV-TK

The EBV-TK gene can be cloned into an expression vector by any meansknown in the art. In particular, EBV-TK gene from the viral strain B-958can be cloned into the vector pCMV as described by Gustafson E. A. etal. Antimicrob. Agents Chemother. 1998, 42(11), 2923-31. The vector canthen be transferred using any known reagent in the art into any desiredviable cell line, for example, using Lipofectamine reagent to transfectthe vector into human cell lines.

VIII Gene Therapy

Eukaryotic cells that may be transduced with vectors (infectious viralparticles or plasmids) containing a gene for the expression of EBV-TKinclude, but are not limited to, primary cells, such as primarynucleated blood cells, such as leukocytes, granulocytes, monocytes,macrophages, lymphocytes (including T-lymphocytes and B-lymphocytes),totipotent stem cells, and tumor infiltrating lymphocytes (TIL cells);bone marrow cells; endothelial cells; epithelial cells; keratinocytes;stem cells; hepatocytes, including hepatocyte precursor cells;hepatocytes, including hepatocyte precursor cells; fibroblasts;mesenchymal cells; mesothelial cells; parenchymal cells, or other cellsof tumor derivation.

In one embodiment, the cells may be targeted to a specific site, wherebythe cells function as a therapeutic at such site. Alternatively, thecells may be cells that are not targeted to a specific site, and suchcells function as a systemic therapeutic.

Transduced cells may be used, for example, in the treatment of abnormalcellular proliferation in a host, and in particular a human, byintroducing to host cells, such as blood cells that specifically“target” to a site of abnormal cellular proliferation (for example atumor), that have been removed from a cancer patient and expanded inculture, transduced with a vector (infectious viral particles orplasmid) in accordance with the present invention which contain genesthat encode EBV-TK. Optionally, the vector can also contain genes thatenhance the anti-tumor effects of the cell. The cells can be expanded innumber before or after transduction with the vector containing thedesired genes. Thus, the procedure is performed in such a manner thatupon injection into the patient, the transformed cells will produceEBV-TK in the patient's body, preferably at the site of the tumoritself.

The gene of the present invention carried by the transduced cellsspecifically comprises the sequence for EBV-TK, but can be also compriseany sequence that directly or indirectly enhances the therapeuticeffects of the cells. The gene carried by the transduced cells can alsoinclude sequences that allow the transduced cells to exert a therapeuticeffect that it would not ordinarily have, such as a gene encoding aclotting factor useful in the treatment of hemophilia. The gene canencode one or more products having therapeutic effects. Examples ofsuitable genes include those that encode cytokines such as TNF, GMCSF,interleukins (interleukins 1-18), interferons (alpha, beta,gamma-interferons), T-cell receptor proteins and Fc receptors forantigen-binding domains of antibodies, such as immunoglobulins.Additional examples of suitable genes include genes that modify cells to“target” to a site in the body to which the cells would not ordinarilytarget, thereby making possible the use of the cell's therapeuticproperties at that site. For example, blood cells such as TIL cells canbe modified, for example, by introducing a Fab portion of a monoclonalantibody into the cells, thereby enabling the cells to recognize achosen antigen. Likewise, blood cells having therapeutic properties canbe used to target, for example, a tumor, that the blood cells would notnormally target. Other genes useful in cancer therapy can be used toencode chemotacetic factors that cause an inflammatory response at aspecific site, thereby having a therapeutic effect. Other examples ofsuitable genes include genes encoding soluble CD4 that is used in thetreatment of AIDS and genes encoding alpha-1-antitrypsin, which isuseful in the treatment of emphysema caused by alpha-1-antitrypsindeficiency.

IX Gene Therapy Vectors

In general, a gene cannot be directly inserted into a cell. It must bedelivered to the cell using a carrier known as a “vector.” The mostcommon types of vectors used in gene therapy are viruses. Scientists useviruses because they have a unique ability to attach to or enter acell's DNA. Viruses used as vectors in gene therapy are geneticallydisabled; they are unable to reproduce themselves, though they canreplicate coordinately with the cellular DNA. Many gene therapy clinicaltrials rely on mouse retroviruses to deliver the desired gene. Otherviruses used as vectors include adenoviruses, adeno-associated viruses,poxviruses and the herpes virus.

For example, cells from the patient are removed and grown in thelaboratory. The cells are exposed to the virus that is carrying thedesired gene. The virus enters the cells, and the desired gene becomespart of the cells' DNA. The cells grow in the laboratory and are thenreturned to the patient. This type of gene therapy is called ex vivo,which means “outside the body.” The gene is transferred into thepatient's cells while the cells are outside the patient's body. In otherstudies, vectors (viral, bacterial) or liposomes (fatty particles) areused to deliver the desired gene to cells in the patient's body. Thisform of gene therapy is called in vivo, because the gene is transferredto cells inside the patient's body.

When these gene delivery vectors are used to carry genes into the body,they might alter more than the intended cells. Another danger is thatthe new gene might be inserted in the wrong location in the DNA,possibly causing cancer or other damage. In addition, when using in vivogene delivery systems, there is a chance that the DNA could beintroduced into reproductive cells, producing inheritable changes.

Other concerns include the possibility that transferred genes could be“overexpressed,” producing so much of a protein as to be harmful; that apathogen vector could cause inflammation or an immune reaction; and inthe case where a virus is used as the vector, it could be transmittedfrom the patient to other individuals or into the environment.

There are many vectors known in the art. Any known vector can be used inthe present invention. In a preferred embodiment of the presentinvention, the vector can target a specific cell type for specific genedelivery.

Adenoviral Vectors

Any of the adenoviral vectors can be used to transfect cells and/or celllines with EBV-TK. Adenoviruses are non-enveloped viruses containing alinear double stranded DNA genome. While there are over 40 serotypestrains of adenovirus, most of which cause benign respiratory tractinfections in humans, subgroup C serotypes 2 or 5 are predominantly usedas vectors. The life cycle does not normally involve integration intothe host genome, rather they replicate as episomal elements in thenucleus of the host cell and consequently there is no risk ofinsertional mutagenesis. The wild type adenovirus genome isapproximately 35 kb of which up to 30 kb can be replaced with foreignDNA (Smith A. E. (1995) Viral vectors in gene therapy. Annual Review ofMicrobiology 49: 807-838; Verma I. M. & Somia N. (1997) Genetherapy—promises, problems and prospects. Nature 389: 239-242). Thereare four early transcriptional units (E1, E2, E3 and E4) that haveregulatory functions, and a late transcript, which codes for structuralproteins. Progenitor vectors have either the E1 or E3 gene inactivated,with the missing gene being supplied in trans either by a helper virus,plasmid or integrated into a helper cell genome (human fetal kidneycells, line 293; Graham F. L., Smiley J., Russell W. L., Nairn R. (1997)Characterization of a human cell line transformation by DNA fromadenovirus 5. Gen. Virol. 36: 59-72). Second generation vectorsadditionally use an E2a temperature sensitive mutant (Engelhardt J. F.,Litsky L., Wilson, J. M. (1994) Prolonged gene expression in cotton ratlung with recombinant adenoviruses defective in E2a. Human Gene Therapy5: 1217-1229) or an E4 deletion (Armentano D., Zabner J., Sacks C.,Sookdeo C. C., Smith M. P., St. George J. A., Wadsworth S. C., Smith A.E., Gregory R. J. (1997) Effect of the E4 region on the presistance oftransgene expression from adenovirus vectors. J. Virol. 71: 2408-2416).The most recent “gutless” vectors contain only the inverted terminalrepeats (ITRs) and a packaging sequence around the transgene, all thenecessary viral genes being provided in trans by a helper virus (ChenH., Mack L. M., Kelly R., Ontell M., Kochanek S., Clemens P. R. (1997)Persistence in muscle of an adenoviral vector that lacks all viralgenes. Proc. Natl. Acad. Sci. USA 94: 1645-1650).

Adenoviral vestors are very efficient at transducing target cells invitro and vivo, and can be produced at high titres (>10¹¹/mL). With theexception of Geddes et al. (Geddes B. J., Harding T. C., Lightman S. L.,Uney J. B. (1997) Long term gene therapy in the CNS: Reversal ofhypothalamic diabetes insipidus in the Brattleboro rat by using anadenovirus expressing arginine vasopressin. Nature Medicine 3:1402-1404), who showed prolonged transgene expression in rat brainsusing an E1 deletion vector, transgene expression in vivo fromprogenitor vectors is typically transient (Verma I. M. & Somia N. (1997)Gene therapy—promises, problems and prospects. Nature 389: 239-242).Following intravenous injection, 90% of the administered vector isdegraded in the liver by a non-immune mediated mechanism (Worgall S.,Wolff G., Falck-Pedersen E., Crystal R. G. (1997) Innate immunemechanisms dominate elimination of adenoviral vectors following in vivoadministration. Human Gene Therapy 8: 37-44). Thereafter, an MHC class Irestricted immune response occurs, using CD8+ CTLs to eliminate virusinfected cells and CD4+ cells to secrete IFN-alpha which results inanti-adenoviral antibody (Yang Y., Wilson J. M. (1995) Clearance ofadenovirus-infected hepatocytes by MHC class I restricted CD4+ CTLs invivo. J. Immunol. 155: 2564-2569). Alteration of the adenoviral vectorcan remove some CTL epitopes, however the epitopes recognized differwith the host MHC haplotype (Sparer T. E., Wynn S. G., Clark D. J.,Kaplan J. M., Cardoza L. M., Wadsworth S. C., Smith A. E., Gooding L. R.(1997) Generation of cytotoxic T lymphocytes against immunorecessiveepitopes after multiple immunizations with adenovirus vectors isdependent on haplotype. J. Virol. 71: 2277-2284; Jooss K., Ertl H. C.J., Wilson J. M. (1998) Cytotoxic T-lymphocyte target proteins and theirhistocompatibility complex class I restriction in response to adenovirusvectors delivered to mouse liver. J. Virol. 72: 2945-2954). Theremaining vectors, in those cells that are not destroyed, have theirpromoter inactivated (Armentano D., Zabner J., Sacks C., Sookdeo C. C.,Smith M. P., St. George J. A., Wadsworth S. C., Smith A. E., Gregory R.J. (1997) Effect of the E4 region on the persistence of transgeneexpression from adenovirus vectors. J. Virol. 71: 2408-2416) andpersisting antibody prevents subsequent administration of the vector.

Approaches to avoid the immune response involving transientimmunosuppressive therapies have been successful in prolonging transgeneexpression and achieving secondary gene transfer (Jooss K., Yang Y.,Wilson J. M. (1996) Cyclophosphamide diminishes inflammation andprolongs expression following delivery of adenoviral vectors to mouseliver and lung. Human Gene Therapy 7: 1555-1566; Kay M. A., Meuse L.,Gown A. M., Linsley P., Hollenbaugh D., Aruffo A., Ochs H. D., Wilson C.B. (1997) Transient immunomodulation with anti-CD40 ligand and CTLA41genhances presistance and secondary adenovirus-mediated gene transferinto mouse liver. Proc. Natl. Acad. Sci. USA 94: 4686-4691). A lessinterventionist method has been to induce oral tolerance by feeding thehost UV inactivated vector (Kagami H., Atkinson J. C., Michalek S. M.,Handelman B., Yu S., Baum B. J., O'Connell B. (1998) Repetitiveadenovirus administration to the parotid gland: role of immunologicalbarriers and induction of oral tolerance. Human Gene Therapy 9:305-313). However, it is desirable to manipulate the vector rather thanthe host. Although only replication-deficient vectors are used, viralproteins are expressed at a very low level, which are presented to theimmune system. The development of vectors containing fewer genes,culminating in the “gutless” vectors which contain no viral codingsequences, has resulted in prolonged in vivo transgene expression inliver tissue (Schiedner G., Morral N., Parks R. J., Wu Y., Koopmans S.C., Langston C., Graham F. L., Beaudet A. L., Kochanek S. (1998) GenomicDNA transfer with a high-capacity adenovirus vector results in improvedin vivo gene expression and decreased toxicity. Nature Genetics 18:180-183). The initial delivery of large amounts of DNA packaged withinadenovirus proteins, the majority of which will be degraded andpresented to the immune system may still cause problems for clinicaltrials. Moreover the human population is heterogeneous with respect toMHC haplotype and a proportion of the population will have been alreadyexposed to the adenoviral strain (Gahry-Sdard H., Molinier-Frenkel V.,Le Boulaire C., Saulnier P., Opolon P., Lengange R., Gautier E., LeCesne A., Zitvogel L., Venet A., Schatz C., Courtney M., Le ChevalierT., Tursz T., Guillet J., Farace F. (1997) Phase I trial of recombinantadenovirus gene transfer in lung cancer. J. Clin. Invest. 100:2218-2226).

Until recently, the mechanism by which the adenovirus targeted the hostcell was poorly understood. Tissue specific expression was thereforeonly possible by using cellular promoter/enhancers, such as the myosinlight chain 1 promoter (Shi Q., Wang Y., Worton R. (1997) Modulation ofthe specificity and activity of a cellular promoter in an adenoviralvector. Human Gene Therapy 8: 403-410) or the smooth muscle cell SM22apromoter (Kim S., Lin H., Barr E., Chu L., Leiden J. M., Parrnacek M. S.(1997) Transcriptional targeting of replication-defective adenovirustransgene expression to smooth muscle cells in vivo. J. Clin. Invest.100: 1006-1014), or by direct delivery to a local area (Rome J. J.,Shayani V., Newman K. D., Farrell S., Lee S. W., Virmani R., Dicheck D.A. (1994) Adenoviral vector mediated gene transfer into sheep arteriesusing a double-balloon catheter. Human Gene Therapy 5: 1249-1258).Uptake of the adenovirus particle has been shown to be a two stageprocess involving an initial interaction of a fiber coat protein in theadenovirus with a cellular receptor or receptors, which include the MHCclass I molecule (Hong S. S., Karayan L., Toumier J., Curiel D. T.,Boulanger P. A. (1997) Adenovirus type 5 fiber knob binds to MHC class Ia2 domain at the surface of human epithelial and B lymphoblastoid cells.EMBO J. 16: 2294-2306) and the Coxsackie virus-adenovirus receptor(Bergelson J. M., Cunningham J. A., Droguett G., Kurt-Jones A. E.,Krithivas A., Hong J. S., Horwitz M. S., Crowell R. L., Finberg R. W.(1997) Isolation of a common receptor for Coxsackie virus B viruses andadenoviruses 2 and 5. Science 275: 1320-1323). The penton base proteinof the adenovirus particle then binds to the integrin family of cellsurface heterodimers (Wickham T. J., Mathias P., Cheresh D. A., NemerowG. R. (1993) Integrins avb3 and avb5 promote adenovirus internalizationbut not virus attachment. Cell 73: 309-319) allowing internalization viareceptor mediated endocytosis. Most cells express primary receptors forthe adenovirus fiber coat protein, however internalization is moreselective (Harris J. D. & Lemoine N. R. (1996) Strategies for targetedgene therapy. Trends in Genetics 12: 400-404). Methods of increasingviral uptake include stimulating the target cells to express anappropriate integrin (Davison E., Diaz R. M., Hart I. R., Santis G.,Marshall J. F. (1997) Integrin a5b1-mediated adenovirus infection isenhanced by the integrin-activating antibody TS2/16. Journal of Virology71: 6204-6207) and conjugating an antibody with specificity for thetarget cell type to the adenovirus (Wickham T. J., Lee G. M, Titus J.A., Titus J. A., Sconocchia G., Bakacs T., Kovesdi I., Segal D. M.(1997b). Targeted adenovirus-mediated gene delivery to T cells via CD3.J. Virol. 71: 7663-7669; Goldman C. K., Rogers B. E., Douglas J. T.,Sonsowski B. A., Ying W., Siegal G. P., Baird A., Campain J. A., CurielD. T. (1997) Targeted gene delivery to Kaposi's sarcoma cells via thefibroblast growth factor receptor. Cancer Res. 57: 1447-1451). The useof antibodies though increases the production difficulties of the vectorand the potential risk of activating the complement system. Byincorporating receptor binding motifs into the fiber coat protein(Wickham T. J., Tzeng E., Shears II, L. L., Roelvink P. W., Li Y., LeeG. M., Brough D. E., Lizonova A., Kovesdi I. (1997a) Increased in vitroand in vivo gene transfer by adenovirus vectors containing chimericfiber proteins. J. Virol. 71: 8221-8229) were able to redirect the virusto bind the integrin expressed by damaged endothelial or smooth musclecells, or heparin sulfate receptors, which are expressed by many celltypes.

Adeno-Associated Viral Vectors

Any of the adeno-associated viral vectors can be used to transfect cellsand/or cell lines with EBV-TK or KHSV-TK. Adeno-associated viruses (AAV)are non-pathogenic human parvoviruses, dependant on a helper virus,usually adenovirus, to proliferate. They are capable of infecting bothdividing and non dividing cells, and in the absence of a helper virusintegrate at a specific point of the human host genome(19q13.fwdarw.qter) at a high frequency (Kotin R. M., Siniscalco M.,Samulski R. J., Zhu X. D., Hunter L., Laughlin C. A., McLaughlin S.,Muzyczka N., Rocchi M., Bems K. I. (1990) Site-specific integration byadeno-associated virus. Proc. Natl. Acad. Sci. USA 87: 2211-2215). Thewild type genome is a single stranded DNA molecule, consisting of twogenes; rep, coding for proteins which control viral replication,structural gene expression and integration into the host genome, andcap, which codes for capsid structural proteins. At either end of thegenome is a 145 bp terminal repeat (TR), containing a promoter.

When used as a vector, the rep and cap genes are replaced by thetransgene and its associated regulatory sequences. The total length ofthe insert cannot greatly exceed 4.7 kb, the length of the wild typegenome (Smith A. E. (1995) Viral vectors in gene therapy. Ann. Rev.Microbiol. 49: 807-838). Production of the recombinant vector requiresthat rep and cap are provided in trans, along with helper virus geneproducts (E1a, E1b, E2a, E4 and VA RNA from the adenovirus genome). Theconventional method is to cotransfect two plasmids, one for the vectorand another for rep and cap, into 293 cells infected with adenovirus(Samulski R. J., Chang L., Shenk T. (1989) Helper free stocks ofrecombinant adeno-associated viruses: normal integration does notrequire viral gene expression. J. Virol. 63: 3822-3828). This method,however, is cumbersome, low yielding (<10⁴ particles/ml) and prone tocontamination with adenovirus and wild type AAV. One of the reasons forthe low yield is the inhibitory effect of the rep gene product onadenovirus replication (Vincent K. A, Piraino S. T., Wadsworth S. C.(1997) Analysis of recombinant adeno-associated virus packaging andrequirements for rep and cap gene products. J. Virol. 71: 1897-1905).More recent protocols remove all adenoviral structural genes and use represistant plasmids (Xiao X., Li J., Samulski R. J. (1998) Production ofhigh-titer recombinant adeno-associated virus vectors in the absence ofhelper adenovirus. J. Virol. 72: 2224-2232) or conjugate a repexpression plasmid to the mature virus prior to infection (Fisher K. J.,Kelley W. M, Burda J. F., Wilson J. M. (1996) A noveladenovirus-adeno-associated virus hybrid vector that displays efficientrescue and delivery of the AAV genome. Human Gene Therapy 7: 2079-2087).

In the absence of rep, the AAV vector will only integrate at random, asa single provirus or head to tail concatamers, once the terminal repeatshave been slightly degraded (Rutledge E. A. & Russell D. W. (1997)Adeno-associated virus vector integration junctions. J. Virol. 71:8429-8436). Interest in AAV vectors has been due to their integrationinto the host genome allowing prolonged transgene expression. Genetransfer into vascular epithelial cells (Maeda Y., Ikeda U., OgasawaraY., Urabe M., Takizawa T., Saito T., Colosi P., Kurtzman G., Shimada K.,Ozawa, K. (1997) Gene transfer into vascular cells usingadeno-associated virus (AAV) vectors. Cardiovascular Res. 35: 514-521),striated muscle (Fisher K. J., Jooss K., Alston J., Yang Y., Haecker S.E., High K., Pathak R., Raper S. E., Wilson J. M. (1997) Recombinantadeno-associated virus for muscle directed gene delivery. NatureMedicine 3: 306-316; Herzog R. W., Hagstrom J. N., Kung S., Tai S. J.,Wilson J. M., Fisher K. J., High K. A. (1997) Stable gene transfer andexpression of human blood coagulation factor IX after intramuscularinjection of recombinant adeno-associated virus. Proc. Natl. Acad. Sci.USA 94: 5804-5809) and hepatic cells (Snyder R. O., Miao C. H., PatijnG. A., Spratt S. K., Danos O., Nagy D., Gown A. M., Winter B., Meuse L.,Cohen L. K., Thompson A. R., Kay, M. A. (1997) Persistent andtherapeutic concentrations of human factor IX in mice after hepatic genetransfer of recombinant AAV vectors. Nature Genetics 16: 270-275) hasbeen reported, with prolonged expression when the transgene is notderived from a different species. Neutralizing antibody to the AAVcapsid may be detectable, but does not prevent readministration of thevector or shut down promoter activity. It is possibly due to thesimplicity of the viral capsid, that the immune response is so muted. AsAAV antibodies will be present in the human population this will requirefurther investigation. There has been no attempt to target particularcell types other than by localized vector delivery.

In particular, the adeno-associated vectors disclosed in U.S. Pat. No.5,693,531, which is hereby incorporated by reference, can be used,including AAVp5neo; pSV-β-galactosidase; TRF169; LZ11; pSP72;pSP72mLacZ; pAdRSV4; pAdRSVnLacZ; AAVmLac; SV40; pBluescriptSK; pSV40ori AAV1; and pKMT11.

Retroviral Vectors

Any of the retroviral vectors can be used to transfect cells and/or celllines with EBV-TK. Retroviruses are a class of enveloped virusescontaining a single stranded RNA molecule as the genome. Followinginfection, the viral genome is reverse transcribed into double strandedDNA, which integrates into the host genome and is expressed as proteins.The viral genome is approximately 10 kb, containing at least threegenes: gag (encoding core proteins), pol (encoding reversetranscriptase) and env (encoding the viral envelope protein). At eachend of the genome are long terminal repeats (LTRs) which includepromoter/enhancer regions and sequences involved with integration. Inaddition there are sequences required for packaging the viral DNA (psi)and RNA splice sites in the env gene. Some retroviruses containproto-oncogenes, which when mutated can cause cancers; however, in theproduction of vectors these are removed. Retroviruses can also transformcells by integrating near a cellular proto-oncogene and drivinginappropriate expression from the LTR, or by disrupting a tumorsuppresser gene. Such as event, termed insertional mutagenesis, thoughextremely rare, could still occur when retroviruses are used as vectors.

Retroviral vectors are most frequently based upon the Moloney murineleukemia virus (Mo-MLV), which is an amphotrophic virus, capable ofinfecting both mouse cells, enabling vector development in mouse models,and human cells, enabling human treatment. The viral genes (gag, pol andenv) are replaced with the transgene of interest and expressed fromplasmids in the packaging cell line. Because the non-essential geneslack the packaging sequence (psi) they are not included in the virionparticle. To prevent recombination resulting in replication-competentretroviruses, all regions of homology with the vector backbone should beremoved and non-essential genes should be expressed in at least twotranscriptional units (Markowitz D., Goff S., Bank A. (1988) A safepackaging line for gene transfer: separating viral genes on twodifferent plasmids. J. Virol. 62: 1120-1124). Even so,replication-competent retroviruses do arise at a low frequency.

The essential regions include the 5′- and 3′-LTRs, and the packagingsequence lying downstream of the 5′-LTR. Transgene expression can eitherbe driven by the promoter/enhancer region in the 5′-LTR, or byalternative viral (e.g., cytomegalovirus, Rous sarcoma virus) orcellular (e.g., beta actin, tyrosine) promoters. Mutational analysis hasshown that up to the entire gag coding sequence and the immediateupstream region can be removed without effecting viral packaging ortransgene expression (Kim S. H., Yu S. S., Park J. S, Robbins P. D, AnC. S., Kim S. (1998) Construction of retroviral vectors with improvedsafety, gene expression, and versatility. J. Virol. 72: 994-1004).However the exact positioning of the transgene start codon and smallalterations of the 5′-LTR influence transgene expression (Rivire I.,Brose K., Mulligan R. C. (1995) Effects of retroviral vector design onexpression of human adenosine deaminase in murine bone marrow transplantrecipients engrafted with genetically modified cells. Proc. Natl. Acad.Sci. USA 92: 6733-6737). To aid identification of transformed cellsselectable markers, such as neomycin and beta-galactosidase, can beincluded and transgene expression can be improved by the addition ofinternal ribosome-binding sites (Saleh M. (1997) A retroviral vectorthat allows co-expression of two genes and the versatility of alternateselection markers. Human Gene Therapy 8: 979-983). The availablecarrying capacity for retroviral vectors is approximately 7.5 kb (VermaI. M. & Somia N. (1997) Gene therapy-promises, problems and prospects.Nature 389: 239-242), which is too small for some genes, even if thecDNA is used.

The retroviral envelope interacts with a specific cellular protein todetermine the target cell range. Altering the env gene or its producthas proved a successful means of manipulating the cell range. Approacheshave included direct modifications of the binding site between theenvelope protein and the cellular receptor, however these approachestend to interfere with subsequent internalization of the viral particle(Harris J. D. & Lemoine N. R. (1996) Strategies for targeted genetherapy. Trends in Genetics 12: 400-404). By replacing a portion of theenv gene with 150 codons from the erythropoietin protein (EPO), Kasaharaet al. (Kasahara N., Dozy A. M., Kan Y. W. (1994) Tissue-specifictargeting of retroviral ligand-receptor interactions. Science 266:1374-1376) were able to target EPO receptor bearing cells with highaffinity. Coupling an antibody to the viral particle with affinity for asecond cell specific antibody via a streptavodin bridge, improves viraluptake, but internalization tends to lead to viral degradation (Roux P.,Jeanteur P., Piechaczyk M. (1989) A versatile and potentially generalapproach to the targeting of specific cell types by means of majorhistocompatibility complex class I and class II antigens by mouseecotropic murine leukemia virus-derived viruses. Proc. Natl. Acad. Sci.USA 86: 9079-9083). Neda et al. (Neda H., Wu C. H., Wu G. Y. (1991)Chemical modification of an ecotropic murine leukemia virus results inredirection of its target cell specificity. J. Biol. Chem. 266:14143-14146) treated viral particles with lactose, which resulted inuptake by cells, principally hepatocytes, expressing asialoglycoproteinreceptors. Subsequently, there was efficient viral transgene expression,possibly due to acidification of the endosome, allowing fusion of theviral envelope with the endosome membrane.

Viruses differ with respect to their tropisms; therefore, by replacingthe env gene with that of another virus, the host range can be extended,in a technique known as pseudotyping. Vesicular stomatitis virus Gprotein has been included in Mo-MLV derived vectors (Burns J. C.,Matsubara T., Lozinski G., Yee J., Freidmann T., Washabaugh C. H.,Tsonis P. A. (1994) Pantropic retroviral vector-mediated gene transfer,integration, and expression in cultured newt limb cells. Dev. Biol. 165:285-289), which are also more stable when purified byultracentrifugation. Recently, Qing et al. (Qing K., Bachelot T.,Mukherjee P., Wang X., Peng L., Yoder M. C., Leboulch P., Srivastava A.(1997) Adeno-associated virus type 2-mediated transfer of ecotropicretrovirus receptor cDNA allows ecotropic retroviral transduction ofestablished and primary human cells. J. Virol. 71: 5663-5667) improvedtransduction into numerous cell lines by first treating the recipientcells with an adeno-associated vector (discussed below) expressing thecellular receptor for retroviral envelope protein.

A requirement for retroviral integration and expression of viral genesis that the target cells should be dividing. This limits gene therapy toproliferating cells in vivo or ex vivo, whereby cells are removed fromthe body, treated to stimulate replication and then transduced with theretroviral vector, before being returned to the patient. When treatingcancers in vivo, tumor cells are preferentially targeted (Roth J. A.,Nguyen D., Lawrence D. D., Kemp B. L., Carrasco C. H., Ferson D. Z.,Hong W. K., Komaki R., Lee J. J., Nesbitt J. C., Pisters K. M. W.,Putnam J. B., Schea R., Shin D. M., Walsh G. L., Dolormente M., Han C.I., Martin F. D., Yen N., Xu K., Stephens L. C., McDonnell T. J.,Mukhopadhyay T., Cai D. (1996). Retrovirus mediated wild-type p53 genetransfer to tumors of patients with lung cancer. Nature Medicine 2:985-991; Tait D. L., Obermiller P. S., Redlin-Frazier S., Jensen R. A.,Welcsh P., Dann J., King M, Johnson D. H., Holt J. T. (1997). A phase Itrial of retroviral BRCA1sv gene therapy in ovarian cancer. Clin. CancerRes. 3: 1959-1968). However, ex vivo cells can be more efficientlytransduced, because of exposure to higher virus titers and growthfactors (Glimm H., Kiem H. P., Darovsky B., Storb R., Wolf J., Diehl V.,Mertelsmann R., Kalle C. V. (1997). Efficient gene transfer in primitiveCD34+/CD381o human bone marrow cells reselected after long term exposureto GALV-pseudotyped retroviral vector. Human Gene Therapy 8: 2079-2086).Furthermore ex vivo-treated tumor cells will associate with the tumormass and can direct tumoricidal effects (Oldfield E. H. & Ram Z. (1995)Intrathecal gene therapy for the treatment of leptomeningealcarcinomatosis. Human Gene Therapy 6: 55-85; Abdel-Wahab Z., Weltz C.,Hester D., Pickett N., Vervaert C., Barber J. R., Jolly D., Seigler H.F. (1997) A phase I clinical trial of immunotherapy withinterferon-gamma gene-modified autologous melanoma cells. Cancer 80:401-412).

Though transgene expression is usually adequate in vitro and initiallyin vivo, prolonged expression is difficult to attain. Retroviruses areinactivated by cl complement protein and an anti-alpha galactosylepitope antibody, both present in human sera (Rother P. R., William L.F., Springhom J. P., Birks W. C., Sandrin M. S., Squinto S. P., RollinsS. A. (1995) A novel mechanism of retrovirus inactivation in human serummediated by anti-a-galactosyl natural antibody. J. Exp. Med. 182:1345-1355; Rollins S. A., Birks C. W., Setter E., Squinto S,P., RotherR. P. (1996) Retroviral vector producer cell killing in human serum ismediated by natural antibody and complement: strategies for evading thehumoral immune response. Human Gene Therapy 7: 619-626). Transgeneexpression is also reduced by inflammatory interferons, specificallyIFN-alpha and IFN-gamma acting on viral LTRs (Ghazizadeh S., Carroll J.M., Taichman L. B. (1997) Repression of retrovirus-mediated transgeneexpression by interferons: implications for gene therapy. J. Virol. 71:9163-9169). As the retroviral genome integrates into the host genome, itis most likely that the viral LTR promoters are inactivated; therefore,one approach has been to use promoters for host cell genes, such astyrosine (Diaz R. M., Eisen T., Hart I. R., Vile R. G. (1998) Exchangeof viral promoter/enhancer elements with regulatory sequences generatedtargeted hybrid long terminal repeat vectors for gene therapy ofmelanoma. J. Virol. 72: 789-795). Clearly this is an area wherecontinued research is needed.

Lentiviruses are a subclass of retroviruses that are able to infect bothproliferating and non-proliferating cells. They are considerably morecomplicated than simple retroviruses, containing an additional sixproteins, tat, rev, vpr, vpu, nef and vif. Current packaging cell lineshave separate plasmids for a pseudotype env gene, a transgene construct,and a packaging construct supplying the structural and regulatory genesin trans (Naldini L., Blmer U., Gallay P., Ory D., Mulligan R., Gage F.H., Verma I. M., Trono D. (1996) In vivo gene delivery and stabletransduction of non-dividing cells by a lentiviral vector. Science 272:263-267). Early results using marker genes have been promising, showingprolonged in vivo expression in muscle, liver and neuronal tissue (BlmerU., Naldini L., Kafri T., Trono D., Verma I. M., Gage F. H. (1997)Highly efficient and sustained gene transfer in adult neurons with alentivirus vector. J. Virol. 71: 6641-6649; Miyoshi H., Takahashi M.,Gage F. H., Verma I. M. (1997) Stable and efficient gene transfer intothe retina using an HIV-based lentiviral vector. Proc. Natl. Acad. Sci.USA 94: 10319-10323; Kafri T., Blmer U., Peterson D. A., Gage F. H.,Verma I. M. (1997) Sustained expression of genes delivered into liverand muscle by lentiviral vectors. Nature Genetics 17: 314-317).Interestingly the transgenes are driven by an internally engineeredcytomegalovirus promoter, which unlike the situation in MoMLV vectors,is not inactivated. This may be due to the limited inflammatory responseto vector injection, which was equal in magnitude to the saline control(Blmer U., Naldini L., Kafri T., Trono D., Verma I. M., Gage F. H.(1997) Highly efficient and sustained gene transfer in adult neuronswith a lentivirus vector. J. Virol. 71: 6641-6649).

The lentiviral vectors used are derived from the human immunodeficiencyvirus (HIV) and are being evaluated for safety, with a view to removingsome of the non-essential regulatory genes. Mutants of vpr and vif areable to infect neurons with reduced efficiency, but not muscle or livercells (Blmer U., Naldini L., Kafri T., Trono D., Verma I. M., Gage F. H.(1997) Highly efficient and sustained gene transfer in adult neuronswith a lentivirus vector. J. Virol. 71: 6641-6649; Kafri T., Blmer U.,Peterson D. A., Gage F. H., Verma I. M. (1997) Sustained expression ofgenes delivered into liver and muscle by lentiviral vectors. NatureGenetics 17: 314-317).

In a particular embodiment, the retroviral vectors pLXIN, pSIR, pLXSH,pLNCX, pLAPSN, pFB and pFB-Neo are used.

Herpes Simplex Viral Vectors

Any of the herpes simplex viral vectors can be used to transfect cellsand/or cell lines with EBV-TK. Herpes simplex virus type 1 (HSV-1) is ahuman neurotropic virus; consequently interest has largely focused onusing HSV-1 as a vector for gene transfer to the nervous system.Wild-type HSV-1 virus is able to infect neurons and either proceed intoa lytic life cycle or persist as an intranuclear episome in a latentstate. Latently infected neurons function normally and are not rejectedby the immune system. Though the latent virus is transcriptionallyalmost silent, it does possess neuron-specific promoters that arecapable of functioning during latency. Antibodies to HSV-1 are common inthe human population; however complications due to herpes infection,such as encephalitis, are very rare.

The viral genome is a linear, double-stranded DNA molecule of 152 kb.There are two unique regions, long and short (termed UL and US) whichare linked in either orientation by internal repeat sequences (IRL andIRS). At the non-linker end of the unique regions are terminal repeats(TRL and TRS). There are up to 81 genes (Marconi P., Krisky D., OliginoT., Poliani P. L., Ramakrishnan R., Goins W. F., Fink D. A., Glorioso J.C. (1996) Replication-defective herpes simplex virus vectors for genetransfer in vivo. Proc. Natl. Acad. Sci. USA 93: 11319-11320), of whichabout half are not essential for growth in cell culture. Once thesenon-essential genes have been deleted, 40-50 kb of foreign DNA can beaccommodated within the virus (Glorioso J. C., DeLuca N. A., Fink D. J.(1995) Development and application of herpes simplex virus vectors forhuman gene therapy. Annual Review of Microbiology 49: 675-710). Threemain classes of HSV-1 genes have been identified: the immediate-early(IE or alpha) genes, early (E or beta) genes, and late (L or gamma)genes.

Following infection in susceptible cells, lytic replication is regulatedby a temporally coordinated sequence of gene transcription. Vmw65 (ategument structural protein) activates the immediate early genes (IPO,ICP4, ICP22, ICP27 and ICP477) that are transactivating factors allowingthe production of early genes. The early genes encode proteins fornucleotide metabolism and DNA replication. Late genes are activated bythe early genes and encode structural proteins. The entire cycle takesless than 10 h and invariably results in cell death.

The molecular events leading to the establishment of latency have notbeen fully determined. Gene expression during latency is driven by thelatency associated transcripts (LATS) located in the IRL region of thegenome. Two LATs (2.0 and 1.5 kb) are transcribed in the oppositedirection to the IE gene ICPO. LATs have a role in HSV-1 reactivationfrom latency (Steiner I., Spivack J. G., Lirette R. P., Brown S. M.,MacLean A. R., Subak-Sharpe J. H., Fraser N. W. (1989) Herpes simplexvirus type 1 latency associated transcripts are evidently not essentialfor latent infection. EMBO Journal 8: 505-511) and the establishment oflatency (Sawtell N. M. & Thompson R. L. (1992) Herpes simplex virus type1 latency-associated transcription unit promotes anatomicalsite-dependant establishment and reactivation from latency. J. Virol.66: 2157-2169). Two latency active promoters that drive expression ofthe LATs have been identified (Marconi P., Krisky D., Oligino T.,Poliani P. L., Ramakrishnan R., Goins W. F., Fink D. A., Glorioso J. C.(1996) Replication-defective herpes simplex virus vectors for genetransfer in vivo. Proc. Natl. Acad. Sci. USA 93: 11319-11320) and mayprove useful for vector transgene expression.

Two basic approaches have been used for production of HSV-1 vectors,namely amplicons and recombinant HSV-1 viruses. Amplicons arebacterially produced plasmids containing col E1 ori (an Escherichia coliorigin of replication), OriS (the HSV-1 origin of replication), HSV-1packaging sequence, the transgene under control of an immediate-earlypromoter and a selectable marker (Federoff H. J., Geschwind M. D.,Geller A. I., Kessler J. A. (1992) Expression of nerve factor in vivofrom a defective herpes simplex virus 1 vector prevents effects ofaxotomy on sympathetic ganglia. Proc. Natl. Acad. Sci. USA 89:1636-1640). The amplicon is transfected into a cell line containing ahelper virus (a temperature sensitive mutant), which provides all themissing structural and regulatory genes in trans. Both the helper- andamplicon-containing viral particles are delivered to the recipient. Morerecent amplicons include an Epstein-Barr virus-derived sequence forplasmid episomal maintenance (Wang S. & Vos J. (1996) A hybridherpesvirus infectious vector based on Epstein-Barr virus and herpessimplex virus type 1 for gene transfer into human cells in vitro and invivo. J. Virol. 70: 8422-8430).

Recombinant viruses are made replication-deficient by deletion of one ofthe immediate-early genes (e.g., ICP4), which is provided in trans.Though they are less pathogenic and can direct transgene expression inbrain tissue, they are toxic to neurons in culture (Marconi P., KriskyD., Oligino T., Poliani P. L., Ramakrishnan R., Goins W. F., Fink D. A.,Glorioso J. C. (1996) Replication-defective herpes simplex virus vectorsfor gene transfer in vivo. Proc. Natl. Acad. Sci. USA 93: 11319-11320).Deletion of a number of immediate-early genes substantially reducescytotoxicity and also allows expression from promoters that would besilenced in the wild-type latent virus. These promoters may be of use indirecting long term gene expression.

Replication-conditional mutants are only able to replicate in certaincell lines. Permissive cell lines are all proliferating and supply acellular enzyme to complement for a viral deficiency. Mutants includethymidine kinase (During M. J., Naegele J. R., O'Malley K. L., Geller A.I. (1994) Long-term behavioral recovery in Parkinsonian rats by an HSVvector expressing tyrosine hydroxylase. Science 266: 1399-1403),ribonuclease reductase (Kramm C. M., Chase M., Herrlinger U., Jacobs A.,Pechan P. A., Rainov N. G., Sena-esteves M., Aghi M., Barnett F. H.,Chiocca E. A., Breakefield X. O. (1997) Therapeutic efficiency andsafety of a second-generation replication-conditional HSV1 vector forbrain tumor gene therapy. Human Gene Therapy 8: 2057-2068), UTPase, orthe neurovirulence factor g34.5 (Kesari S., Randazzo B. P., Valyi-NagyT., Huang Q. S., Brown S. M., MacLean A. R., Lee V. M., Trojanowski J.Q., Fraser N. W. (1995) Therapy of experimental human brain tumors usinga neuroattenuated herpes simplex virus mutant. Lab. Invest. 73:636-648). These mutants are particularly useful for the treatment ofcancers, killing neoplastic cells, which proliferate faster than othercell types (Andreansky S. S., He B., Gillespie G. Y., Soroceanu L.,Market J., Chou J., Roizman B., Whitley R. J. (1996) The application ofgenetically engineered herpes simplex viruses to the treatment ofexperimental brain tumors. Proc. Natl. Acad. Sci. USA 93: 11313-11318;Andreansky S. S., Sorcoceanu L., Flotte E. R., Chou J., Markert J. M.,Gillespie G. Y., Roizman B., Whitley R. J. (1997) Evaluation ofgenetically engineered herpes simplex virus as oncolytic agents forhuman malignant brain tumors. Cancer Res. 57: 1502-1509).

A number of neurological diseases could be amenable to gene therapy byHSV-1 vectors (Kennedy P. G. E. (1997) Potential uses of herpes simplexvirus (HSV) vectors for gene therapy of neurological disorders. Brain120: 1245-1259). Though most attention has focused on cancers, there hasbeen some success in Parkinson's disease by expressing tyrosinehydroxylase in striatal cells (Geller A. I., During M. J., Oh J. Y.,Freese F., O'Malley K. (1995) An HSV-1 vector expressing tyrosinehydroxylase causes production and release of L-DOPA from cultured ratstriatal cells. J. Neurochem. 64: 487-496; During M. J., Naegele J. R.,O'Malley K. L., Geller A. I. (1994) Long-term behavioral recovery inParkinsonian rats by an HSV vector expressing tyrosine hydroxylase.Science 266: 1399-1403), thus replacing the supply of L-dopa. Federoffet al. (Federoff H. J., Geschwind M. D., Geller A. I., Kessler J. A.(1992) Expression of nerve factor in vivo from a defective herpessimplex virus 1 vector prevents effects of axotomy on sympatheticganglia. Proc. Natl. Acad. Sci. USA 89: 1636-1640) induced nerve repairfollowing axotomy of the superior cervical ganglion, by injection of avector expressing nerve growth factor. This resulted in restored levelsof tyrosine hydroxylase.

However, Wood et al. (Wood M. J. A., Byrnes A. P., Pfaff D. W., RabkinS. D., Charlton H. M. (1994) Inflammatory effects of gene-transfer intothe CNS with defective HSV-1 vectors. Gene Therapy 1: 283-291) observedstrong inflammatory responses to HSV-1 amplicon vectors, both at theprimary site of the injection and at secondary sites supplied by nervefibers originating from area of the injection. In addition up to 20% ofexperimental animals may die shortly after injection with HSV-1 vector(Kucharczuk J. C., Randazzo B., Chang M. Y., Amin K. M., Elshami A. A.,Sterman D. H., Rizk N. P., Molnar-Kimber K. L., Brown S. M., MacLean A.R., Litzky L. A., Fraser N. W., Albelda S. M., Kaiser L. R. (1997) Useof a replication-restricted herpes virus to treat experimental humanmalignant mesothelioma. Cancer Research 57: 466-471), though the reasonis unknown. A viral protein, ICP47, has been identified, which reducesviral antigen presentation and may be employed in future HSV-1 vectorsto reduce cytotoxicity (York I. A., Roop C., Andrews D. W., Riddell R.,Graham F. L., Johnson D. C. (1994) A cytosolic herpes simplex virusprotein inhibits antigen presentation to CD8+ T lymphocytes. Cell 77:525-535).

Because of its tropism for neuronal tissue issues of cellular targetinghave been largely overlooked. However, wild type HSV-1 can infect andlyse other non-neuronal cell types, such as skin cells (Al-Saadi S. A.,Clements G. B., Subak-Sharpe J. H. (1983) Viral genes modify herpessimplex virus latency both in mouse footpad and sensory ganglia. J. Gen.Virol. 64: 1175-1179), and it would be advantageous to target a specificsubset of neurons. As HSV-1 will travel down the length of nerveefficient cellular targeting would improve its safety profile when usedas a vector. Indeed a replication-restricted HSV-1 vector has been usedto treat human malignant mesothelioma (Kucharczuk J. C., Randazzo B.,Chang M. Y., Amin K. M., Elshami A. A., Sterman D. H., Rizk N. P.,Molnar-Kimber K. L., Brown S. M., MacLean A. R., Litzky L. A., Fraser N.W., Albelda S. M., Kaiser L. R. (1997) Use of a replication-restrictedherpes virus to treat experimental human malignant mesothelioma. CancerRes. 57: 466-471).

Pox Virus Vectors

Pox virus vectors can also be used (e.g., Yaba-like disease virus: analternative replicating poxvirus vector for cancer gene therapy. Hu Y,Lee J, McCart J A, Xu H, Moss B, Alexander H R, Bartlett D L J Virol2001 75:10300-8).

Non-Viral Vectors

Viral vectors all induce some degree of immunological response and mayhave other safety risks, such as insertional mutagenesis and directtoxicity. Furthermore, large-scale production may be difficult toachieve. Therefore, in some embodiments of the invention, non-viralmethods of gene transfer are used, which may require only a small numberof proteins, have a virtually infinite capacity, have no infectious ormutagenic capability, and large-scale production is possible usingpharmaceutical techniques. There are at least three methods of non-viralDNA transfer, including naked DNA, liposomes and molecular conjugates.

Naked DNA (in the form of a plasmid) can be directly injected intomuscle cells (Wolff J. A., Malone R. W., Williams P., Chong W., AcsadiG., Jani A., Felgner P. L. (1990) Direct gene transfer into mouse musclein vivo. Science 247: 1465-1468) or attached to gold particles that arebombarded into the tissue (Cheng L., Ziegelhoffer P. R., Yang N. S.(1993) In vivo promoter activity and transgene expression in mammaliansomatic tissues evaluated by using particle bombardment. Proc. Natl.Acad. Sci. USA 90: 4455-4459). Though not very efficient, this canresult in prolonged, low-level expression in vivo. The simplicity ofthis method and sustained expression has led to the development of DNAvaccines. Compared to conventional attenuated and protein-basedvaccines, they are unaffected by pre-existing immunity, such as that dueto maternal antibodies, relatively cheap, and can deliver severalpathogen antigens on a single plasmid (Manickan E., Karem K. L., RouseB. T. (1997) DNA vaccines-a modern gimmick or a boon to vaccinology.Critical Reviews in Immunology 17: 139-154). DNA vaccines are beingdeveloped for those pathogens where there is no existing vaccine, suchas HIV (Lekutis C., Shiver J. W., Liu M. A., Letvin L. N. (1997) HIV1env DNA vaccine administered to rhesus monkeys elicits MHC classII-restricted CD4+ T helper cells that secrete IFN-gamma and TNF-alpha.J. Immunol. 158: 4471-4477), or where the current vaccine is not fullyeffective, such as influenza (Macklin M. D., McCabe D., McGregor M. W.,Neumann V., Meyer T., Callan R., Hinshaw V. S., Swain W. S (1998)Immunization of pigs with a particle mediated vaccine to influenza Avirus protects against challenge with homologous virus. J. Virol. 72:1491-1496). By using a highly conserved gene Ulmer et al. (Ulmer J. B.,Donnelly J. J., Parker S. E., Rhodes G. H., Felgner P. L., Dwarki J. J.,Gromkowski S. H., Deck R., DeWitt C. M., Friedman A., Hawe L. A.,Laender K. R., Martinz D., Perry H. C., Shiver J., Montgomery D. L., LiuM. A. (1993) Heterologous protection against influenza by injection ofDNA encoding a viral protein. Science 254: 1745-1749) were able toimmunize mice against two serologically distinct influenza virusstrains. In most cases, however, DNA vaccines have not been shown to bebetter than the existing vaccines (Macklin M. D., McCabe D., McGregor M.W., Neumann V., Meyer T., Callan R., Hinshaw V. S., Swain W. S. (1998)Immunization of pigs with a particle-mediated vaccine to influenza Avirus protects against challenge with homologous virus. J. Virol. 72:1491-1496). The actual type of immune response can be controlled bycotransformation of a gene encoding an appropriate cytokine (Xiang Z. &Ertl H. C. (1995) Manipulation of the immune response to aplasmid-encoded viral antigen by co-inoculation with plasmids expressingcytokines. Immunity 2: 129-135) and this method may prove useful inredirecting inappropriate immune responses (Manickan E., Karem K. L.,Rouse B. T. (1997) DNA vaccines-a modern gimmick or a boon tovaccinology. Critical Reviews in Immunology 17: 139-154). Other uses fornaked DNA include cancer immunopotentiation (discussed below; see alsoCorr M., Tighe H., Lee D., Dudler J., Trieu M., Brinson D. C., Carson D.A. (1997) Costimulation provided by DNA immunization enhances antitumorimmunity. J. Lab. Invest. 159: 4999-5004), repair of pancreatic insulinfunction (Goldfine I. D., German M. S., Tseng H., Wang J., Bolaffi J.L., Chen J., Olson D. C., Rothman S. S. (1997). The endocrine secretionof human insulin and growth hormone by exocrine glands of thegastrointestinal tract. Nature Biotech. 15: 1378-1382), and stimulationof collateral blood vessel development (Takeshita S., Tsurumi Y.,Couffinahl T., Asahara T., Bauters C., Symes J., Ferrara N., Isner J. M.(1996) Gene transfer of naked DNA encoding for three isoforms ofvascular endothelial growth factor endothelial growth factor stimulatescollateral development in vivo. Lab. Invest. 75: 487-501). Expression ofthe gene product in muscle tissue can be improved by thecoadministration of collagenase, papaverine and ischemic conditions(Budker V., Zhang G., Danko I., Williams P., Wolff J. (1998) Theefficient expression of intravascularly delivered DNA in rat muscle.Gene Therapy 5: 272-276). The use of a muscle specific promoter (SkarliM., Kiri A., Vrbova G., Lee C. A., Goldspink G. (1998) Myosin regulatoryelements as vectors for gene transfer by intramuscular injection. GeneTherapy 5: 514-520) and other intragene regulatory sequences, such asthe poly A and transcription termination sequence (Hartikka J., SawdeyM., Conefert-Jensen F., Margalith M., Barnhardt K., Nolasco M., VahlsingH. L., Meek J., Marquet M., Hobart P., Norman J., Manthorpe M. (1996) Animproved plasmid DNA expression vector for direct injection intoskeletal muscle. Human Gene Therapy 7: 1205-1217) will also improvetransgene expression.

Liposomes are lipid bilayers entrapping a fraction of aqueous fluid. DNAwill spontaneously associate to the external surface of cationicliposomes by virtue of its charge, and these liposomes will interactwith the cell membrane (Feigner J. H., Kumar R., Sridhar C. N., WheelerC. J., Tasi Y. J., Border R., Ramsey P., Martin M., Feigner P. L. (1994)Enhanced gene delivery system and mechanism studies with a novel seriesof cationic lipid formulations. J. Biol. Chem. 269: 2550-2561). In vitroup to 90% of certain cell lines may be transfected. By including a smallamount of an anionic lipid in an otherwise cationic liposome, the DNAcan be incorporated into the internal surface of the liposome,protecting it from enzymatic degradation (Alio S. F. (1997) Long termexpression of the human alpha-1-antitrypsin gene in mice employinganionic and cationic liposome vector. Biochem. Pharmacol. 54: 9-13). Tofacilitate uptake into the cell as endosomes, targeting proteins havebeen included in liposomes, such as an anti-MHC antibody (Wang C. &Huang L. (1987) pH-sensitive immunoliposomes mediatetarget-cell-specific delivery and controlled expression of a foreigngene in mouse. Proc. Natl. Acad. Sci. USA 84: 7851-7855), transferrin(Stavridis J. C., Deliconstantinos G., Psallidopoulos M. C., ArmenakasN. S., Hadjiminas D. J., Hadjiminas J. (1986) Construction oftransferrin-coated liposomes for in vivo transport of exogenous DNA tobone marrow erythroblasts in rabbits. Experimental Cell Research 164:568-572), and the Sendai virus or its F protein (Dzau J. V., Mann M. J,Morishita R., Kaneda Y. (1996). Fusigenic viral liposome for genetherapy in cardiovascular disease. Proc. Natl. Acad. Sci. USA 93:11421-11425). The Sendai virus additionally allows the plasmid DNA toescape from the endosome into the cytoplasm, thus avoiding degradation.The inclusion of a DNA-binding protein (e.g., 28 kDa high mobility group1 protein) enhances transcription by bringing the plasmid into thenucleus (Dzau J. V., Mann M. J, Morishita R., Kaneda Y. (1996).Fusigenic viral liposome for gene therapy in cardiovascular disease.Proc. Natl. Acad. Sci. USA 93: 11421-11425). Further proposedimprovements include incorporating the Epstein-Barr virus Ori p andEBNA1 genes in the plasmid (as described above for HSV-1 amplicons) tomaintain the plasmid as an episomal element (Alio S. F. (1997) Long termexpression of the human alpha-1-antitrypsin gene in mice employinganionic and cationic liposome vector. Biochem. Pharmacol. 54: 9-13).

Molecular conjugates consist of protein or synthetic ligands to which aDNA binding agent has been attached. Delivery to the cell can beimproved by using similar techniques to those for liposomes. Targetingproteins include asialoglycoprotein (Wagner E., Cotten M., Foisner R.,Birnstiel M. L. (1991) Transferrin-polycation-DNA complexes: the effectof polycations on the structure of the complex and DNA delivery tocells. Proc. Natl. Acad. Sci. USA 88: 4255-4259), transferrin (Wu C. H.,Wilson J. M., Wu. G. Y. (1989) Targeting genes: delivery and persistentexpression of a foreign gene driven by mammalian regulatory elements invivo. J. Biol. Chem. 264: 16985-16987), polymeric IgA (Ferkol T.,Kaetzel C. S., Davis P. B. (1993) Gene transfer into respiratoryepithelial cells by targeting the polymeric immunoglobulin receptor. J.Clin. Invest. 92: 2394-2400) and adenovirus (Madon J. & Blum H. E.(1996) Receptor mediated delivery of hepatitis B virus DNA and antisenseoligodeoxynucleotides to avian liver cells. Hepatology 24: 474-481).Transgene expression tends to be transient and is limited byendosome/lysosomal degradation.

X Assays and Kits

In one embodiment of the present invention, the transfected cells can beused to create two assay systems to assess the ability of EBV-TK tosensitize cells to candidate active compounds. Cells that expressKHSV-TK can be prepared similarly for assay purposes.

The present invention is described by way of illustration, in thefollowing examples. It will be understood by one of ordinary skill inthe art that these examples are in no way limiting and that variationsof detail can be made without departing from the spirit and scope of thepresent invention.

EXAMPLES

Nucleoside Syntheses

All reagents were used as received unless stated otherwise. Anhydroussolvents were purchased from Aldrich Chemical Company, Inc. (Milwaukee).Melting points (mp) were determined on an Electrothermal digit meltingpoint apparatus and are uncorrected. ¹H and ¹³C NMR spectra wereobtained using a Varian Unity Plus 400 spectrometer at room temperatureand reported in ppm downfield from internal tetramethylsilane. Deuteriumexchange, decoupling experiments or 2D-COSY were performed to confirmproton assignments. Signal multiplicities are represented by s(singlet), d (doublet), dd (doublet of doublets), t (triplet), q(quadruplet), br (broad), bs (broad singlet), m (multiplet). AllJ-values are in Hz. Mass spectra were recorded using a JEOLJMS-SX/SX102A/E mass spectrometer. Elemental analyses were performed byAtlantic Microlab Inc. (Norcross, Ga.). Analytic TLC was performed onWhatman LK6F silica gel plates, and preparative TLC on Whatman PK5Fsilica gel plates. Column chromatography was carried out on Silica Gel(Fisher, S733-1) at atmospheric pressure.

Example 1 β-D-5-E-(2-Carbomethoxyvinyl)-2′-deoxyuridine (2)

To a solution of Pd(OAc)₂ (96 mg, 0.42 mmol) and Ph₃P (240 mg, 0.92mmol) in 1,4-dioxane (90 mL) was added 2′-deoxy-5-iodouridine (1, 2.68g, 7.56 mmol), followed by methyl acrylate (1.6 mL, 19.78 mmol). Themixture was heated at reflux under nitrogen atmosphere for 16 h. The hotmixture was filtered through a pad of celite, and the celite was rinsedwith hot 1,4-dioxane. The combined filtrate was concentrated in vacuo,and the residue was purified by flash chromatography on silica geleluting with CH₂Cl₂/MeOH (85:15 to 75:25) to give the title compound 2as a pale yellow solid (2.17 g, 92%). ¹H NMR (DMSO-d₆) δ 8.42 (s, 1H,H-6), 7.37, 6.85 (2d, J=16 Hz, 2H, CH═CH), 6.12 (t, J=6.4 Hz, 1H, H-1′),5.28 (d, J=4.4 Hz, 1H, OH), 5.19 (t, J=5.2 Hz, 1H, OH), 4.25 (m, 1H,H-3′), 3.81-3.78 (m, 1H, H-4′), 3.68 (s, 3H, CH₃), 3.66-3.56 (m, 2H,H-5′), 2.19-2.15 (m, 2H, H-2′).

In an analogous manner to the preparation of compound 2,β-L-5-E-(2-carbomethoxy-vinyl)-2′-deoxyuridine 7 was prepared fromβ-L-2′-deoxy-5-iodouridine (6; prepared according to Holy A. (1972)Nucleic acid components and their analogues. CLIII. Preparation of2′-deoxy-L-ribonucleosides of the pyrimidine series, Coll. Czech. Chem.Commun. 37, 4072-4086; Lin T.-S., Luo M.-Z., Liu M.-C. (1995) Synthesisof several pyrimidine L-nucleoside analogues as potential antiviralagents, Tetrahedron, 51, 1055-1068). ¹H NMR (DMSO-d₆) δ 8.42 (s, 1H,H-6), 7.37, 6.85 (2d, 2H, J=16 Hz, CH═CH), 6.12 (t, J=6.4 Hz, 1H, H-1′),5.25 (d, J=4.4 Hz, 1H, OH), 5.20 (t, J=5.2 Hz, 1H, OH), 4.25 (m, 1H,H-3′), 3.78-3.81 (m, 1H, H-4′), 3.68 (s, 3H, CH₃), 3.66-3.56 (m, 2H,H-5′), 2.19-2.15 (m, 2H, H-2′).

In an analogous manner to the preparation of compound 2,1-(β-L-arabinofuranosyl)-5-E-(2-carbomethoxyvinyl)uracil 11 was preparedfrom 1-(β-L-arabinofuranosyl)-5-iodouracil (10; prepared according toHoly A. (1972) Nucleic acid components and their analogues. CLIII.Preparation of 2′-deoxy-L-ribonucleosides of the pyrimidine series,Coll. Czech. Chem. Commun. 37, 4072-4086; T.-S. Lin, M.-Z. Luo, M.-C.Liu, (1995) Synthesis of several pyrimidine L-nucleoside analogues aspotential antiviral agents, Tetrahedron, 51, 1055-1068). ¹H NMR(DMSO-d₆) δ 11.5 (s, 1H, NH), 8.22 (s, 1H, H-6), 7.39, 6.85 (2d, J=16Hz, 2H, CH═CH), 6.01 (d, 1H, H-1′), 5.58 (d, 1H, OH), 5.47 (d, 1H, OH),5.23 (t, 1H, OH), 4.15-3.90 (m, 2H, H-2′, H-3′), 3.60-3.70 (m, 3H, H-4′,H-5′), 3.35 (s, 3H, CH₃).

In an analogous manner to the preparation of compound 2,1-(β-D-arabinofuranosyl)-5-E-(2-carbomethoxyvinyl)uracil 17 was preparedfrom 16. ¹NMR (DMSO-d₆) δ 11.5 (s, 1H, NH), 8.22 (s, 1H, H-6), 7.39,6.85 (2d, 2H, J=16 Hz, CH═CH), 6.01 (d, 1H, H-1′), 5.60 (d, 1H, OH),5.48 (d, 1H, OH), 5.24 (t, 1H, OH), 4.15-3.90 (m, 2H, H-2′, H-3′),3.60-3.70 (m, 3H, H-4′, H-5′), 3.35 (s, 3H, CH₃).

In an analogous manner to the preparation of compound 2,5-E-(2-carbomethoxyvinyl)-1-[(1,3-dihydroxy-2-propoxy)methyl]uracil 52was prepared from 46. ¹H NMR (DMSO-d₆) δ 11.5 (s, 1H, NH), 8.40 (s, 1H,H-6), 7.38, 6.85 (2d, 2H, J=16 Hz, CH═CH), 7.10, 6.50 (2br, 2H, 20H),5.15 (s, 2H, CH₂N), 4.60 (m, 2H, CH₂O), 3.69 (s, 3H, CH₃), 3.52 (m, 1H,CHO), 3.41 (m, 2H, 2 CH₂O).

In an analogous manner to the preparation of compound 2,(±)-(1′β,3′α,4′β)-5-E-(2-carbomethoxyvinyl)-1-[3-hydroxy-4-(hydroxymethyl)cyclopentyl]uracil73 was prepared from(±)-(1′β,3′α,4′β)-1-[3-hydroxy-4-(hydroxymethyl)cyclopentyl]-5-iodouracil(72). ¹H NMR (DMSO-d₆) δ 8.40 (s, 1H, H-6), 7.37, 6.85 (2d, 2H, J=16 Hz,CH═CH), 5.0-4.5 (m, 3H, CHN, 20H), 4.00 (m, 1H, CHOH), 3.70 (s, 3H,CH₃), 3.42 (m, 2H, CH₂OH), 2.25-1.05 (m, 5H).

Compound 72 was prepared according to published procedures from(±)-(1β,2α,3α,4β)-4-amino-2,3-dihydroxy-1-cyclop-entanemethanol (71):Shealy Y. F., O'Dell C. A. (1976) J. Heterocycl. Chem. 13, 1015;Herdewijn P., De Clercq E., Balzarini J., Vanderhaeghe H. (1985)Synthesis and antiviral activity of the carbocyclic analogues(E)-5-(2-halovinyl)-2′-deoxyuridines and(E)-5-(2-halovinyl)-2′-deoxycytidines, J. Med. Chem. 28, 550-555.

Compound 71 was prepared according to Kam B. L. & Oppenheimer N. J.(1981) Carbocyclic sugar amines: synthesis and stereochemistry ofracemic α- and β-carbocyclic ribofuranosylamine, carbocycliclyxofuranosylamine, and related compounds, J. Org. Chem. 46, 3268-3272.

In an analogous manner to the preparation of compound 2,(2R,4R)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]-5-E-(2-carbomethoxy-vinyl)uracil100 was prepared from 99. ¹NMR (DMSO-d₆) δ 11.5 (s, 1H, NH), 7.61 (s,1H, H-6), 7.36, 6.85 (2d, 2H, CH═CH), 6.25 (m, 1H, H-2′), 5.22 (t, 1H,OH), 5.10 (m, 1H, H-4′), 4.25-4.05 (m, 4H, H-5′, H-6′), 3.67 (s, 3H,CH₃).

In an analogous manner to the preparation of compound 2,(2S,4S)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]-5-E-(2-carbomethoxy-vinyl)uracil108 was prepared from 107. ¹H NMR (DMSO-d₆) δ 11.5 (s, 1H, NH), 7.61 (s,1H, H-6), 7.36, 6.85 (2d, 2H, CH═CH), 6.25 (m, 1H, H-2′), 5.21 (t, 1H,OH), 5.10 (m, 1H, H-4′), 4.25-4.05 (m, 4H, H-5′, H-6′), 3.67 (s, 3H,CH₃).

Example 2 β-2′-Deoxy-5-E-(2-chlorovinyl)uridine (4)

A solution of 2 (1.25 g, 4.0 mmol) in aqueous NaOH (1 N, 50 mL) wasstirred at room temperature overnight. The solution was cooled to 0° C.and brought to pH 2 by the addition of 4 N HCl. The mixture was stand inice for 30 minutes, and the white precipitate was filtered. The filtratewas evaporated to dryness in vacuo and water was added. The resultingprecipitate was filtered and washed with water. The combined precipitatewas dissolved in water (40 mL) by heating with KOAc (1.96 g, 20 mmol),and N-chlorosuccinimide (536 mg, 4.0 mmol) was added portion-wise. Thesolution was heated at reflux for 3 h, and then concentrated in vacuo.The residue was purified by flash chromatography on silica gel elutingwith CH₂Cl₂/MeOH (9:1) to give the title compound 3 as a white solid(438 mg, 38%). ¹H NMR (DMSO-d₆) δ 11.6 (s, 1H, NH), 8.06 (s, 1H, H-6),7.18, 6.59 (2d, 2H, J=13.2 Hz, CH═CH), 6.13 (t, J=6.4 Hz, 1H, H-1′),5.28 (d, J=4.0 Hz, 1H, OH-3′), 5.12 (t, J=5.2 Hz, 1H, OH-5′), 4.24 (m,1H, H-3′), 3.78 (m, 1H, H-4′), 3.63-3.57 (m, 2H, H-5′), 2.13 (m, 2H,H-2′).

In an analogous manner to the preparation of compound 4,β-L-S-E-(2-chlorovinyl)-2′-deoxyuridine 9 was prepared from 7. ¹H NMR(DMSO-d₆) δ 11.6 (s, 1H, NH), 8.05 (s, 1H, H-6), 7.17, 6.58 (2d, 2H,J=13.2 Hz, CH═CH), 6.13 (t, J=6.8 Hz, 1H, H-1′), 5.27 (d, J=4.4 Hz, 1H,OH-3′), 5.11 (t, J=5.2 Hz, 1H, OH-5′), 4.24 (m, 1H, H-3′), 3.78 (m, 1H,H-4′), 3.66-3.56 (m, 2H, H-5′), 2.13 (m, 2H, H-2′).

In an analogous manner to the preparation of compound 4,1-(β-L-arabinofuranosyl)-5-E-(2-chlorovinyl)uracil 13 was prepared from11. ¹NMR (DMSO-d₆) δ 11.4 (s, 1H, NH), 7.87 (s, 1H, H-6), 7.15, 6.58(2d, J=13 Hz, 2H, CH═CH), 6.01 (d, 1H, H-1′), 5.56 (d, 1H, OH), 5.45 (d,1H, OH), 5.05 (t, 1H, OH), 4.05 (m, 1H, H-2′), 3.90 (m, 1H, H-3′), 3.75(m, 1H, H-4′), 3.61 (m, 2H, H-5′).

In an analogous manner to the preparation of compound 4,1-(β-D-arabinofuranosyl)-5-E-(2-chlorovinyl)uracil 19 was prepared from17. ¹H NMR (DMSO-d₆) δ 11.45 (s, 1H, NH), 7.87 (s, 1H, H-6), 7.15, 6.58(2d, J=13 Hz, 2H, CH═CH), 6.01 (d, 1H, H-1′), 5.57 (d, 1H, OH), 5.44 (d,1H, OH), 5.05 (t, 1H, OH), 4.05 (m, 1H, H-2′), 3.90 (m, 1H, H-3′), 3.75(m, 1H, H-4′), 3.61 (m, 2H, H-5′).

In an analogous manner to the preparation of compound 4,5-E-(2-chlorovinyl)-1-[(1,3-dihydroxy-2-propoxy)methyl]uracil 53 wasprepared from 52. ¹H NMR (DMSO-d₆) δ 11.4 (s, 1H, NH), 8.03 (s, 1H,H-6), 7.28, 6.85 (2d, 2H, J=13 Hz, CH═CH), 7.10, 6.55 (2br, 2H, 20H),5.18 (s, 2H, CH₂N), 4.60 (m, 2H, CH₂O), 3.50 (m, 1H, CHO), 3.41 (m, 2H,2 CH₂O).

In an analogous manner to the preparation of compound 4,(±)-(1′β,3′α,4′β)-5-E-(2-chlorovinyl)-1-[3-hydroxy-4-(hydroxymethyl)cyclopentyl]uracil74 was prepared from 73. ¹H NMR (DMSO-d₆) δ 11.4 (s, 1H, NH), 7.96 (s,1H, H-6), 7.24, 6.75 (2d, J=13 Hz, 2H, CH═CH), 5.0-4.6 (m, 3H, CHN,20H), 4.01 (m, 1H, CHOH), 3.43 (m, 2H, CH₂OH), 2.25-1.35 (m, 5H).

Example 3 1-(β-D-Arabinofuranosyl)uracil (15)

A mixture of uridine (14; 13.25 g, 54.5 mmol), diphenyl carbonate (15.48g, 72.5 mmol), anhydrous NaHCO₃ (349 mg, 4.15 mmol), andhexamethylphosphoric triamide (50 mL) was heated with stirring at 150°C. under nitrogen for 20 min, and then cooled to room temperature. Themixture was poured into cold water (400 mL), and washed with CHCl₃. Et₃N(25 mL) was added, and the aqueous solution was heated at 70° C. for 5h. The solvent was evaporated in vacuo, and the residue was crystallizedfrom MeOH/water to give the title compound 15 as a white solid (11.13 g,84%). ¹H NMR (DMSO-d₆) δ 11.2 (s, 1H, NH), 7.60 (d, J=7.5 Hz, 1H, H-6),5.95 (d, 1H, H-1′), 5.51 (d, J=7.5 Hz, 1H, H-5), 6.30-5.30 (br, 2H, 2OH), 4.10-3.50 (m, 6H, H-2′, H-3′, H-4′, H-5′, OH).

Example 4 1-(β-D-Arabinofuranosyl)-5-iodouracil (16)

A mixture of 15 (1.2 g, 4.9 mmol), iodine (1.50 g, 5.9 mmol), CHCl₃ (6mL), and 1 N HNO₃ (24 mL) was heated at reflux for 2 hours, and thencooled. The crystalline solid was collected by filtration and washedwith ether to give the title compound 16 as a white solid (1.12 g, 62%).¹H NMR (DMSO-d₆) δ 11.6 (s, 1H, NH), 8.10 (s, 1H, H-6), 5.95 (d, 1H,H-1′), 5.52 (br, 1H, OH), 5.40-5.10 (br, 1H, OH), 4.15-3.90 (m, 2H,H-2′, H-3′), 3.80-3.60 (m, 3H, H-4′, H-5′), 3.60-3.30 (m, 1H, OH).

Example 5 β-D-3,5′-Di-O-benzoyl-2′-deoxy-5-iodouridine (20)

To a solution of 1 (5.56 g, 20 mmol) in anhydrous pyridine (100 mL) at0° C. was added benzoyl chloride (6.185 g, 44 mmol) dropwise. Afteraddition, the reaction solution was allowed to warm to room temperature,then heated at 50° C. for 2 h, and evaporated in vacuo to dryness. Theresidue was treated with CHCl₃ (200 mL), and the organic phase waswashed with 1 NH₂SO₄, water, dried, filtered, and concentrated in vacuo.Column chromatography of the residue (CH₂Cl₂/MeOH, 95:5) gave the titlecompound 20 as a white solid (10.35 g, 92%). ¹H NMR (CDCl₃) δ 8.08-7.43(m, 1H, arom., H-6), 6.39-6.36 (m, 1H, H-1′), 5.64 (d, J=6.8 Hz, 1H,H-3′), 4.77 (d, J=3.2 Hz, 2H, H-5′), 4.59 (m, 1H, H-4′), 2.83-2.78,2.38-2.31 (2m, 2H, H-2′).

In an analogous manner to the preparation of compound 20,β-L-3′,5′-di-O-benzoyl-2′-deoxy-5-iodouridine 23 was prepared from 6. ¹HNMR (CDCl₃) δ 8.08-7.42 (m, 11H, arom., H-6), 6.39-6.37 (m, 1H, H-1′),5.64 (d, J=6.8 Hz, 1H, H-3′), 4.77 (d, J=3 Hz, 2H, H-5′), 4.59 (m, 1H,H-4′), 2.83-2.78, 2.38-2.30 (2m, 2H, H-2′).

In an analogous manner to the preparation of compound 20,5-iodo-1-(2,3,5-tri-O-benzoyl-β-L-arabinofuranosyl)uracil 26 wasprepared from 10. ¹H NMR (DMSO-d₆) δ 11.6 (s, 1H, NH), 8.10-7.40 (m,16H, arom., H-6), 6.20 (d, 1H, H-1′), 5.65 (d, 1H, H-2′), 5.35 (d, 1H,H-3′), 5.24 (m, 1H, H-4′), 4.40-4.20 (m, 2H, H-5′).

In an analogous manner to the preparation of compound 20,5-iodo-1-(2,3,5-tri-O-benzoyl-β-D-arabinofuranosyl)uracil 29 wasprepared from 16. ¹H NMR (DMSO-d₆) δ 11.5 (s, 1H, NH), 8.10-7.40 (m,16H, arom., H-6), 6.20 (d, 1H, H-1′), 5.66 (d, 1H, H-2′), 5.35 (d, 1H,H-3′), 5.25 (m, 1H, H-4′), 4.40-4.20 (m, 2H, H-5′).

In an analogous manner to the preparation of compound 20,β-D-2′,3′,5′-tri-O-benzoyl-5-iodouridine 33 was prepared from 32. ¹NMR(CDCl₃) δ 8.32 (br, 1H, NH), 8.16-7.36 (m, 16H, arom., H-6), 6.37 (d,1H, H-1′), 5.90 (m, 1H, H-2′), 5.75 (t, 1H, H-3′), 4.86-4.83 (m, 1H,H-4′), 4.76-4.70 (m, 2H, H-5′).

Example 6 β-D-3′,5′-Di-O-benzoyl-2-deoxy-5-vinyluridine (21)

To a solution of 20 (562 mg, 1 mmol) in 1-methyl-pyrrolidinone (NMP, 3mL) were added tri(2-furyl)phosphine (9 mg), tris(dibenzylideneacetonyl)bispalladium (9 mg), and tributylvinyltin (350 mg, 1.1 mmol)sequentially. After being stirred at room temperature under nitrogenatmosphere for 3 days, the mixture was diluted with EtOAc (50 mL),washed with water, dried and concentrated. The residue was purified byflash chromatography on silica gel eluting with hexane/EtOAc (6:4) togive the title compound 21 as a brownish-yellow foam (450 mg, 97%). ¹HNMR (DMSO-d₆) δ 8.32-7.50 (m, 11H, arom., H-6), 6.32-6.24 (m, 2H,CH═CH₂, H-1′), 5.92, 5.13 (2dd, 2H, CH═CH₂), 5.69-5.65 (m, 1H, H-3′),4.69-4.59 (m, 3H, H-4′, H-5′), 2.84-2.65 (2m, 2H, H-2′).

In an analogous manner to the preparation of compound 21,β-L-3′,5′-di-O-benzyol-2′-deoxy-5-vinyluridine 24 was prepared from 23.¹H NMR (DMSO-d₆) δ 8.31-7.50 (m, 11H, arom., H-6), 6.32-6.24 (m, 2H,CH═CH₂, H-1′), 5.92, 5.13 (2dd, 2H, CH═CH₂), 5.69-5.65 (m, 1H, H-3′),4.69-4.59 (m, 3H, H-4′, H-5′), 2.84-2.66 (2m, 2H, H-2′).

In an analogous manner to the preparation of compound 21,1-(2,3,5-tri-O-benzoyl-β-L-arabinofuranosyl)-5-vinyluracil 27 wasprepared from 26. ¹H NMR (DMSO-d₆) δ 11.5 (s, 1H, NH), 8.11-7.40 (m,16H, arom., H-6), 6.32-6.23 (m, 2H, CH═CH₂, H-1′), 5.92, 5.13 (2dd, 2H,CH═CH₂), 5.65 (d, 1H, H-2′), 5.35 (d, 1H, H-3′), 5.24 (m, 1H, H-4′),4.40-4.230 (m, 2H, H-5′).

In an analogous manner to the preparation of compound 21,1-(2,3,5-tri-O-benzoyl-β-D-arabinofuranosyl)-5-vinyluracil 30 wasprepared from 29. ¹H NMR (DMSO-d₆) δ 11.55 (s, 1H, NH), 8.12-7.40 (m,16H, arom., H-6), 6.32-6.22 (m, 2H, CH═CH₂, H-1′), 5.92, 5.13 (2dd, 2H,CH═CH₂), 5.64 (d, 1H, H-2′), 5.35 (d, 1H, H-3′), 5.24 (m, 1H, H-4′),4.40-4.230 (m, 2H, H-5′).

In an analogous manner to the preparation of compound 21,β-D-2′,3′,5′-tri-O-benzoyl-5-vinyluridine 34 was prepared from 33. ¹HNMR (CDCl₃) δ 8.36 (bs, 1H, NH), 8.15 (d, 1H, H-6), 8.13-7.36 (m, 15H,arom.), 6.45 (d, 1H, H-1′), 6.02-5.72 (m, 4H, CH═CH₂, H-4′), 5.00 (m,1H, H-3′), 4.88 (m, 1H, H-4′), 4.75-4.67 (m, 2H, H-5′).

In an analogous manner to the preparation of compound 21,1-[[1,3-bis(acetoxy)-2-propoxy]methyl]-5-vinyluracil 48 was preparedfrom 47. ¹H NMR (CDCl₃) δ 8.36 (br, 1H, NH), 7.37 (s, 1H, H-6), 6.40(dd, 1H, CH═CH₂), 6.00, 5.31 (2d, 2H, CH═CH₂), 5.28 (s, 2H, CH₂N), 4.23,4.08 (2m, 5H, 2 CH₂, CHO), 2.04 (s, 6H, 2 CH₃).

In an analogous manner to the preparation of compound 21,β-D-2′-deoxy-3′,5′-di-O-p-toluoyl-4′-thio-5-vinyluridine 56 was preparedfrom 55. ¹H NMR (CDCl₃) δ 7.95-7.25 (m, 8H, arom., H-6), 6.69 (dd, 1H,H-1′), 6.31-6.25 (m, 2H, CH═CH₂, H-1′), 5.90, 5.11 (2dd, 2H, CH═CH₂),5.76 (m, 1H, H-3′), 4.68 (m, 2H, H-5′), 4.05 (m, 1H, H-4′), 2.75, 2.41(2m, 2H, H-2′), 2.42 (s, 6H, 2 CH₃).

In an analogous manner to the preparation of compound 21,1-(4-thio-2,3,5-tri-O-benzyl-β-D-arabinofuranosyl)-5-vinyluracil 64 wasprepared from 63. ¹H NMR (CDCl₃) δ 8.10 (br, 1H, NH), 7.82 (s, 1H, H-6),7.40-7.15 (m, 15H, arom.), 6.32-6.25 (m, 2H, CH═CH₂, H-1′), 5.90, 5.12(2dd, 2H, CH═CH₂), 4.70-4.40 (m, 6H, 3 CH₂), 4.22 (m, 2H, H-2′, H-3′),3.65 (m, 2H, H-5′), 3.40 (m, 1H, H-4′).

In an analogous manner to the preparation of compound 21,(±)-(1′β,3′α,4′β)-1-[3-acetoxy-4-(acetoxymethyl)cyclopentyl]-5-vinyluracil76 was prepared from 75. ¹H NMR (DMSO-d₆) δ 11.5 (bs, 1H, NH), 8.02 (s,1H, H-6), 6.36 (dd, 1H, CH═CH₂), 5.91, 5.12 (2dd, 2H, CH═CH₂), 5.00 (m,1H, CHO), 4.92 (m, 1H, CHN), 4.18, 4.05 (2m, 2H, CH₂O), 2.02 (s, 6H, 2CH₃), 2.45-1.85 (2m, 4H, 2 CH₂), 1.57 (m, 1H, CH).

In an analogous manner to the preparation of compound 21 but replacingtributyl(vinyl)tin with 2-(tributylstannyl)furan,β-D-2′-deoxy-3′,5′-di-O-benzoyl-5-furanyluridine 80 was prepared from20. ¹H NMR (CDCl₃) δ 8.16 (s, 1H, H-6), 8.09-7.41 (m, 10H, arom.), 6.93(d, J=2.8 Hz, 1H, furanyl), 6.82 (d, J=1.2 Hz, 1H, furanyl), 6.56 (m,1H, furanyl), 6.31 (m, 1H, H-1′), 5.71 (d, J=6.4 Hz, 1H, H-3′), 4.78 (d,2H, H-5′), 4.65 (m, 1H, H-4′), 2.85, 2.52 (2m, 2H, H-2′).

In an analogous manner to the preparation of compound 21 but replacingtributyl(vinyl)tin with 2-(tributylstannyl)thiophene,β-D-2′-deoxy-3′,5′-di-O-benzoyl-5-(thien-2-yl)uridine 81 was preparedfrom 20. ¹H NMR (CDCl₃) δ 8.06-7.19 (m, 13H, arom., H-6), 6.86 (t, 1H,J=4.4 Hz, thienyl), 6.49 (m, 1H, H-1′), 5.69 (d, 1H, J=6.4 Hz, H-3′),4.86-4.77 (m, 2H, H-5′), 4.65-4.63 (m, 1H, H-4′), 2.86, 2.44 (2m, 2H,H-2′).

In an analogous manner to the preparation of compound 21 but replacingtributyl(vinyl)tin with 2-(tributylstannyl)furan,β-L-2′-deoxy-3′,5′-di-O-benzoyl-5-(2-furanyl)uridine 87 was preparedfrom 23. ¹H NMR (CDCl₃) δ 8.16 (s, 1H, H-6), 8.10-7.40 (m, 10H, arom.),6.93 (d, J=2.8 Hz, 1H, furanyl), 6.82 (d, J=1.2 Hz, 1H, furanyl), 6.56(m, 1H, furanyl), 6.31 (m, 1H, H-1′), 5.71 (d, J=6.4 Hz, 1H, H-3′), 4.78(d, 2H, H-5′), 4.65 (m, 1H, H-4′), 2.85, 2.52 (2m, 2H, H-2′).

In an analogous manner to the preparation of compound 21 but replacingtributyl(vinyl)tin with 2-(tributylstannyl)thiophene,β-L-2′-deoxy-3′,5′-di-O-benzoyl-5-(thien-2-yl)uridine 88 was preparedfrom 23. ¹H NMR (CDCl₃) δ 8.06-7.19 (m, 13H, arom., H-6), 6.86 (t, 1H,J=4.4 Hz, thienyl), 6.49 (m, 1H, H-1′), 5.69 (d, 1H, J=6.4 Hz, H-3′),4.86-4.77 (m, 2H, H-5′), 4.65-4.63 (m, 1H, H-4′), 2.86, 2.44 (2m, 2H,H-2′).

In an analogous manner to the preparation of compound 21,(2R,4R)-1-[2-(benzyloxymethyl)-1,3-dioxolan-4-yl]-5-vinyluracil 95 wasprepared from 94 (compound 94 was prepared according to J.-S. Lin, T.Kira, E. Gullen, Y. Choi, F. Qu, C. K. Chu, Y.-C. Cheng, J. Med. Chem.1999, 42, 2212-2217). ¹H NMR (CDCl₃) δ 8.15 (br, 1H, NH), 8.20-7.40 (m,6H, arom., H-6), 6.45-6.25 (m, 2H, CH═CH₂, H-2′), 6.03, 5.31 (2dd, 2H,CH═CH₂), 5.20 (m, 1H, H-4′), 4.30-4.10 (m, 4H, H-5′, H-6′).

In an analogous manner to the preparation of compound 21,(2S,4S)-1-[2-(benzyloxymethyl)-1,3-dioxolan-4-yl]-5-vinyluracil 103 wasprepared from 102 (compound 102 was prepared according to the publishedprocedure: J.-S. Lin, T. Kira, E. Gullen, Y. Choi, F. Qu, C. K. Chu,Y.-C. Cheng, J. Med. Chem. 1999, 42, 2212-2217). ¹H NMR (CDCl₃) δ 8.15(br, 1H, NH), 8.20-7.40 (m, 6H, arom., H-6), 6.45-6.25 (m, 2H, CH═CH₂,H-2′), 6.03, 5.31 (2dd, 2H, CH═CH₂), 5.20 (m, 1H, H-4′), 4.30-4.10 (m,4H, H-5′, H-6′).

In an analogous manner to the preparation of compound 21 but replacingtributyl(vinyl)tin with 2-(tributylstannyl)furan,(2R,4R)-1-[2-(benzyloxymethyl)-1,3-dioxolan-4-yl]-5-(2-furanyl)uracil110 was prepared from 94. ¹H NMR (CDCl₃) δ 8.16 (bs, 1H, NH), 8.10-7.41(m, 6H, arom., H-6), 6.92 (d, 1H, furanyl), 6.82 (d, 1H, furanyl), 6.55(m, 1H, furanyl), 6.32 (m, 1H, H-2′), 5.20 (m, 1H, H-4′), 4.30-4.10 (m,4H, H-5′, H-6′).

In an analogous manner to the preparation of compound 21 but replacingtributyl(vinyl)tin with 2-(tributylstannyl)thiophene,(2R,4R)-1-[2-(benzyloxymethyl)-1,3-dioxolan-4-yl]-5-(2-thienyl)uracil111 was prepared from 94. ¹H NMR (CDCl₃) δ 8.06-7.20 (m, 7H, arom.,H-6), 6.85 (t, 1H, thienyl), 6.29 (m, 1H, H-2′), 5.20 (m, 1H, H-4′),4.28-4.10 (m, 4H, H-5′, H-6′).

In an analogous manner to the preparation of compound 21 but replacingtributyl(vinyl)tin with 2-(tributylstannyl)furan,(2S,4S)-1-[2-(benzyloxymethyl)-1,3-dioxolan-4-yl]-5-(2-furanyl)uracil116 was prepared from 102. ¹H NMR (CDCl₃) δ 8.16 (bs, 1H, NH), 8.10-7.41(m, 6H, arom., H-6), 6.92 (d, 1H, furanyl), 6.82 (d, 1H, furanyl), 6.55(m, 1H, furanyl), 6.32 (m, 1H, H-2′), 5.20 (m, 1H, H-4′), 4.30-4.10 (m,4H, H-5′, H-6′).

In an analogous manner to the preparation of compound 21 but replacingtributyl(vinyl)tin with 2-(tributylstannyl)thiophene,(2S,4S)-1-[2-(benzyloxymethyl)-1,3-dioxolan-4-yl]-5-(2-thienyl)uracil117 was prepared from 102. ¹H NMR (CDCl₃) δ 8.06-7.20 (m, 7H, arom.,H-6), 6.85 (t, 1H, thienyl), 6.29 (m, 1H, H-2′), 5.20 (m, 1H, H-4′),4.28-4.10 (m, 4H, H-5′, H-6′).

In an analogous manner to the preparation of compound 21 but replacingtributyl(vinyl)tin with tributyl(phenyl)tin,β-D-2′-deoxy-3′,5′-di-O-benzoyl-5-propynyluridine 127 was prepared from20. ¹H NMR (CDCl₃) δ 8.10-7.45 (m, 11H, arom., H-6), 6.43 (m, 1H, H-1′),5.65 (d, 1H, H-3′), 4.83-4.71 (m, 2H, H-5′), 4.61 (m, 1H, H-4′), 2.79,2.38 (2m, 2H, H-2′), 1.87 (s, 3H, CH₃).

In an analogous manner to the preparation of compound 21 but replacingtributyl(vinyl)tin with tributyl(phenyl)tin,β-D-2′-deoxy-3′,5′-di-O-benzoyl-5-(phenylethynyl)uridine 128 wasprepared from 20. ¹H NMR (CDCl₃) δ 8.09-7.28 (m, 16H, arom., H-6), 6.45(m, 1H, H-1′), 5.67 (d, 1H, H-3′), 4.88-4.76 (m, 2H, H-5′), 4.63 (m, 1H,H-4′), 2.83, 2.40 (2m, 2H, H-2′).

In an analogous manner to the preparation of compound 21 but replacingtributyl(vinyl)tin with tributyl(phenyl)tin,β-D-2′-deoxy-3′,5′-di-O-benzoyl-5-phenyluridine 129 was prepared from20. ¹H NMR (CDCl₃) δ 8.09-7.21 (m, 16H, arom., H-6), 6.38 (m, 1H, H-1′),5.65 (m, 1H, H-3′), 4.77 (m, 2H, H-5′), 4.60 (m, 1H, H-4′), 2.79, 2.32(2m, 2H, H-2′).

In an analogous manner to the preparation of compound 21 but replacingtributyl(vinyl)tin with tributyl(phenyl)tin,β-L-2′-deoxy-3′,5′-di-O-benzoyl-5-phenyluridine 133 was prepared from23. ¹H NMR (CDCl₃) δ 8.09-7.21 (m, 16H, arom., H-6), 6.38 (m, 1H, H-1′),5.65 (m, 1H, H-3′), 4.77 (m, 2H, H-5′), 4.60 (m, 1H, H-4′), 2.79, 2.32(2m, 2H, H-2′).

Example 7 β-D-2′-Deoxy-5-vinyluridine (22)

A solution of 21 (183 mg, 0.4 mmol) in ammonium-MeOH solution (2M, 25mL) was stirred in a stoppered flask at room temperature for 20 h, andthen concentrated in vacuo. Column chromatography of the residue(CH₂Cl₂/MeOH, 9:1) gave the title compound 22 as a white crystallinesolid (88 mg, 87%). ¹H NMR (DMSO-d₆) δ 11.37 (s, 1H, NH), 8.10 (s, 1H,H-6), 6.36 (dd, 1H, CH═CH₂), 6.15 (t, 1H, J=6.4 Hz, H-1′), 5.91, 5.11(2dd, 2H, CH═CH₂), 5.21, 5.09 (2br, 2H, 20H), 4.24 (m, 1H, H-3′), 3.78(m, 1H, H-4′), 3.59 (m, 1H, H-5′), 2.15-2.11 (m, 2H, H-2′).

In an analogous manner to the preparation of compound 22,β-L-2′-deoxy-5-vinyluridine 25 was prepared from 24. ¹H NMR (DMSO-d₆) δ11.4 (s, 1H, NH), 8.10 (s, 1H, H-6), 6.36 (dd, 1H, CH═CH₂), 6.15 (t, 1H,J=6.4 Hz, H-1′), 5.91, 5.11 (2dd, 2H, CH═CH₂), 5.21, 5.10 (2br, 2H,20H), 4.24 (m, 1H, H-3′), 3.78 (m, 1H, H-4′), 3.59 (m, 1H, H-5′),2.15-2.11 (m, 2H, H-2′).

In an analogous manner to the preparation of compound 22,1-(β-L-arabinofuranosyl)-5-vinyluracil 28 was prepared from 27. ¹H NMR(DMSO-d₆) δ 11.42 (s, 1H, NH), 7.89 (s, 1H, H-6), 6.37 (dd, 1H, CH═CH₂),6.01 (d, 1H, H-1′), 5.87, 5.08 (2dd, 2H, CH═CH₂), 5.62 (d, 1H, OH), 5.48(d, 1H, OH), 5.18 (t, 1H, OH), 4.05 (m, 1H, H-2′), 3.93 (m, 1H, H-3′),3.73 (m, 1H, H-4′), 3.65 (m, 2H, H-5′).

In an analogous manner to the preparation of compound 22,1-(β-D-arabinofuranosyl)-5-vinyluracil 31 was prepared from 30. ¹H NMR(DMSO-d₆) δ 11.42 (s, 1H, NH), 7.90 (s, 1H, H-6), 6.37 (dd, 1H, CH═CH₂),6.01 (d, 1H, H-1′), 5.87, 5.08 (2dd, 2H, CH═CH₂), 5.62 (d, 1H, OH), 5.48(d, 1H, OH), 5.18 (t, 1H, OH), 4.05 (m, 1H, H-2′), 3.93 (m, 1H, H-3′),3.73 (m, 1H, H-4′), 3.65 (m, 2H, H-5′).

In an analogous manner to the preparation of compound 22,β-D-5-vinyluridine 35 was prepared from 34. ¹H NMR (DMSO-d₆) δ 11.4 (s,1H, NH), 8.21 (s, 1H, H-6), 6.37 (dd, 1H, CH═CH₂), 5.78 (d, 1H, H-1′),5.92, 5.11 (2dd, 2H, CH═CH₂), 5.44 (d, 1H, OH), 5.25 (d, 1H, OH), 5.10(t, 1H, OH), 4.06 (m, 1H, H-2′), 4.01 (m, 1H, H-3′), 3.86 (m, 1H, H-4′),3.69, 3.60 (2m, 2H, H-5′).

In an analogous manner to the preparation of compound 22,1-[(1,3-dihydroxy-2-propoxy)methyl]-5-vinyluracil 49 was prepared from48. ¹H NMR (DMSO-d₆) δ 11.25 (br, 1H, NH), 7.90 (s, 1H, H-6), 7.20, 6.60(2br, 2H, 20H), 6.36 (dd, 1H, CH═CH₂), 5.95, 5.12 (2d, 2H, CH═CH₂), 5.19(s, 2H, CH 2N), 4.61 (m, 2H, CH₂O), 3.53 (m, 1H, CHO), 3.40 (m, 2H,CH₂O).

In an analogous manner to the preparation of compound 22,(±)-(1′β,3′β,4′β)-1-[3-hydroxy-4-(hydroxymethyl)cyclopentyl]-5-vinyluracil77 was prepared from 76. ¹H NMR (DMSO-d₆) δ 11.5 (bs, 1H, NH), 7.96 (s,1H, H-6), 6.35 (dd, 1H, CH═CH₂), 5.90, 5.10 (2dd, 2H, CH═CH₂), 4.95 (m,1H, CHN), 4.75, 4.63 (2bs, 2H, 20H), 4.00 (m, 1H, CHOH), 3.43 (m, 2H,CH₂OH), 2.25-1.75 (m, 4H, 2 CH₂), 1.40 (m, 1H, CH).

In an analogous manner to the preparation of compound 22,β-D-2′-deoxy-5-(2-furanyl)uridine 82 was prepared from 80. ¹H NMR(DMSO-d₆) δ 11.63 (s, 1H, NH), 8.33 (s, 1H, H-6), 7.60 (s, 1H, furanyl),6.84 (d, J=2.8 Hz, 1H, furanyl), 6.51 (m, 1H, furanyl), 6.20 (t, 1H,J=6.4 Hz, H-1′), 5.29 (d, 1H, J=4.4 Hz, OH-3′), 5.11 (t, 1H, J=4.8 Hz,OH-5′), 4.27 (m, 1H, H-3′), 3.83 (m, 1H, H-4′), 3.60 (m, 2H, H-5′), 2.16(m, 2H, H-2′).

In an analogous manner to the preparation of compound 22,β-D-2′-deoxy-5-(thien-2-yl)uridine 83 was prepared from 81. ¹H NMR(DMSO-d₆) δ 11.7 (s, 1H, NH), 8.57 (s, 1H, H-6), 7.45-7.44 (m, 1H,thienyl), 7.7.39-7.38 (m, 1H, thienyl), 7.05-7.03 (m, 1H, thienyl), 6.21(t, 1H, J=6.4 Hz, H-1′), 5.31-5.27 (m, 2H, 20H), 4.31 (m, 1H, H-3′),3.83 (m, 1H, H-4′), 3.68-3.63 (m, 2H, H-5′), 2.23-2.17 (m, 2H, H-2′).

In an analogous manner to the preparation of compound 22,β-L-2′-deoxy-5-(2-furanyl)uridine 89 was prepared from 87. ¹H NMR(DMSO-d₆) δ 11.63 (s, 1H, NH), 8.33 (s, 1H, H-6), 7.60 (s, 1H, furanyl),6.84 (d, J=2.8 Hz, 1H, furanyl), 6.51 (m, 1H, furanyl), 6.20 (t, 1H,J=6.4 Hz, H-1′), 5.29 (d, 1H, J=4.4 Hz, OH-3′), 5.11 (t, 1H, J=4.8 Hz,OH-5′), 4.27 (m, 1H, H-3′), 3.83 (m, 1H, H-4′), 3.60 (m, 2H, H-5′), 2.16(m, 2H, H-2′).

In an analogous manner to the preparation of compound 22,β-L-2′-deoxy-5-(thien-2-yl)uridine 90 was prepared from 88. ¹H NMR(DMSO-d₆) δ 11.7 (s, 1H, NH), 8.57 (s, 1H, H-6), 7.45-7.44 (m, 1H,thienyl), 7.7.39-7.38 (m, 1H, thienyl), 7.05-7.03 (m, 1H, thienyl), 6.21(t, 1H, J=6.4 Hz, H-1′), 5.31-5.27 (m, 2H, 20H), 4.31 (m, 1H, H-3′),3.83 (m, 1H, H-4′), 3.68-3.63 (m, 2H, H-5′), 2.23-2.17 (m, 2H, H-2′).

In an analogous manner to the preparation of compound 22,(2R,4R)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]-5-vinyluracil 96 wasprepared from 95. ¹H NMR (DMSO-d₆) δ 11.46 (br, 1H, NH), 8.19 (s, 1H,H-6), 6.36 (dd, 1H, CH═CH₂), 6.23 (m, 1H, H-2′), 5.90, 5.11 (2dd, 2H,CH═CH₂), 5.35 (t, 1H, H-4′), 4.93 (t, 1H, OH), 4.15-4.00 (m, 2H, H-5′),3.68 (m, 1H, H-6′).

In an analogous manner to the preparation of compound 22,(2R,4R)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]-5-iodouracil 99 wasprepared from 94. ¹H NMR (DMSO-d₆) δ 11.4 (br, 1H, NH), 8.17 (s, 1H,H-6), 6.22 (m, 1H, H-2′), 5.35 (t, 1H, H-4′), 4.92 (t, 1H, OH),4.15-4.00 (m, 2H, H-5′), 3.68 (m, 1H, H-6′).

In an analogous manner to the preparation of compound 22,(2S,4S)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]-5-vinyluracil 104 wasprepared from 103. ¹H NMR (DMSO-d₆) δ 11.46 (br, 1H, NH), 8.19 (s, 1H,H-6), 6.36 (dd, 1H, CH═CH₂), 6.23 (m, 1H, H-2′), 5.90, 5.11 (2dd, 2H,CH═CH₂), 5.35 (t, 1H, H-4′), 4.93 (t, 1H, OH), 4.15-4.00 (m, 2H, H-5′),3.68 (m, 1H, H-6′).

In an analogous manner to the preparation of compound 22,(2S,4S)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]-5-iodouracil 107 wasprepared from 102. ¹H NMR (DMSO-d₆) δ 11.4 (br, 1H, NH), 8.17 (s, 1H,H-6), 6.22 (m, 1H, H-2′), 5.35 (t, 1H, H-4′), 4.92 (t, 1H, OH),4.15-4.00 (m, 2H, H-5′), 3.68 (m, 1H, H-6′).

In an analogous manner to the preparation of compound 22,(2R,4R)-5-(2-furanyl)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]uracil 112was prepared from 110. ¹H NMR (DMSO-d₆) δ 11.6 (br, 1H, NH), 8.33 (s,1H, H-6), 7.60 (d, 1H, furanyl), 6.84 (d, 1H, furanyl), 6.50 (d, 1H,furanyl), 6.22 (m, 1H, H-2′), 5.35 (t, 1H, H-4′), 4.92 (t, 1H, OH),4.15-4.00 (m, 2H, H-5′), 3.68 (m, 1H, H-6′).

In an analogous manner to the preparation of compound 22,(2R,4R)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]-5-(thien-2-yl)uracil 114was prepared from 111. ¹H NMR (DMSO-d₆) δ 11.67 (br, 1H, NH), 8.57 (s,1H, H-6), 7.44 (dd, 1H, thienyl), 7.38 (dd, 1H, thienyl), 7.04 (dd, 1H,thienyl), 6.22 (m, 1H, H-2′), 5.35 (t, 1H, H-4′), 4.92 (t, 1H, OH),4.15-4.00 (m, 2H, H-5′), 3.68 (m, 1H, H-6′).

In an analogous manner to the preparation of compound 22,(2S,4S)-5-(2-furanyl)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]uracil 118was prepared from 116. ¹H NMR (DMSO-d₆) δ 11.6 (br, 1H, NH), 8.33 (s,1H, H-6), 7.60 (d, 1H, furanyl), 6.84 (d, 1H, furanyl), 6.50 (d, 1H,furanyl), 6.22 (m, 1H, H-2′), 5.35 (t, 1H, H-4′), 4.92 (t, 1H, OH),4.15-4.00 (m, 2H, H-5′), 3.68 (m, 1H, H-6′).

In an analogous manner to the preparation of compound 22,(2S,4S)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]-5-(thien-2-yl)uracil 120was prepared from 117. ¹H NMR (DMSO-d₆) δ 11.67 (br, 1H, NH), 8.57 (s,1H, H-6), 7.44 (dd, 1H, thienyl), 7.38 (dd, 1H, thienyl), 7.04 (dd, 1H,thienyl), 6.22 (m, 1H, H-2′), 5.35 (t, 1H, H-4′), 4.92 (t, 1H, OH),4.15-4.00 (m, 2H, H-5′), 3.68 (m, 1H, H-6′).

In an analogous manner to the preparation of compound 22,β-D-2′-deoxy-5-propynyluridine 130 was prepared from 127. ¹H NMR(DMSO-d₆) δ 11.56 (s, 1H, NH), 8.10 (s, 1H, H-6), 6.10 (t, 1H, J=6.4 Hz,H-1′), 5.23 (d, 1H, J=4.0 Hz, OH-3′), 5.09 (t, 2H, J=6.4 Hz, OH-5′),4.21 (m, 1H, H-3′), 3.77 (m, 1H, H-4′), 3.58 (m, 1H, H-5′), 2.10 (m, 2H,H-2′), 1.97 (s, 3H, CH₃).

In an analogous manner to the preparation of compound 22,β-D-2′-deoxy-5-(phenylethynyl)uridine 131 was prepared from 128. ¹H NMR(DMSO-d₆) δ 11.70 (s, 1H, NH), 8.37 (d, 1H, H-6), 7.47-7.39 (m, 5H,arom.), 6.12 (t, 1H, J=6.4 Hz, H-1′), 5.26 (d, 1H, J 4.0 Hz, OH-3′),5.17 (t, 2H, J=6.4 Hz, OH-5′), 4.24 (m, 1H, H-3′), 3.80 (m, 1H, H-4′),3.63-3.57 (m, 1H, H-5′), 2.15 (m, 2H, H-2′).

In an analogous manner to the preparation of compound 22,β-D-2′-deoxy-5-phenyluridine 132 was prepared from 129. ¹H NMR (DMSO-d₆)δ 11.52 (s, 1H, NH), 8.20 (s, 1H, H-6), 7.55-7.29 (m, 5H, arom.), 6.24(t, 1H, J=6.4 Hz, H-1′), 5.27 (d, 1H, J=4.4 Hz, OH-3′), 5.13 (t, 2H,J=6.4 Hz, OH-5′), 4.28 (m, 1H, H-3′), 3.81 (m, 1H, H-4′), 3.60 (m, 1H,H-5′), 2.24-2.15 (m, 2H, H-2′).

In an analogous manner to the preparation of compound 22,β-L-2′-deoxy-5-phenyluridine 134 was prepared from 133. ¹H NMR (DMSO-d₆)δ 11.52 (s, 1H, NH), 8.20 (s, 1H, H-6), 7.55-7.29 (m, 5H, arom.), 6.24(t, 1H, J=6.4 Hz, H-1′), 5.27 (d, 1H, J=4.4 Hz, OH-3′), 5.13 (t, 2H,J=6.4 Hz, OH-5′), 4.28 (m, 1H, H-3′), 3.81 (m, 1H, H-4′), 3.60 (m, 1H,H-5′), 2.24-2.15 (m, 2H, H-2′).

Example 8β-2′-Deoxy-3′,5′-di-O-benzoyl-5-[(2-trimethylsilyl)ethynyl]uridine (36)

To a solution of 20 (2.81 g, 5 mmol), PdCl₂ (89 mg, 0.5 mmol) Ph₃P (261mg, 1 mmol) and CuI (95 mg, 0.5 mmol) in THF (50 mL) was added Et₃N(1.01 g, 10 mmol, 1.39 mL), followed by trimethylsilylacetylene (737 mg,7.5 mmol). The mixture was heated at 40° C. for 4 h under argon, andthen evaporated to dryness. The residue was purified by flashchromatography on silica gel eluting with hexane/EtOAc (9:1) to give thetitle compound 36 as a white solid (2.08 g, 78%). ¹H NMR (CDCl₃) δ 8.31(s, 1H, H-6), 8.10-7.43 (m, 10H, arom.), 6.39-6.36 (dd, 1H, H-1′), 5.60(m, 1H, H-3′), 4.83 (dd, 1H, H-4′), 4.67-4.57 (m, 2H, H-5′), 2.81-2.76,2.30-2.27 (2m, 2H, H-2′), 0.14 (s, 9H, 3 CH₃).

In an analogous manner to the preparation of compound 36,β-L-2′-deoxy-3′,5′-di-O-benzoyl-5-[(2-trimethylsilyl)ethynyl]uridine 38was prepared from 23. ¹H NMR (CDCl₃) δ 8.31 (s, 1H, H-6), 8.10-7.42 (m,10H, arom.), 6.40-6.35 (dd, 1H, H-1′), 5.60 (m, 1H, H-3′), 4.83 (dd, 1H,H-4′), 4.67-4.57 (m, 2H, H-5′), 2.81-2.76, 2.30-2.27 (2m, 2H, H-2′),0.14 (s, 9H, 3 CH₃).

In an analogous manner to the preparation of compound 36,1-(2′,3′,5′-tri-O-benzoyl-β-L-arabinofuranosyl)-5-[(2-trimethylsilyl)ethynyl]uracil40 was prepared from 26. ¹H NMR (DMSO-d₆) δ 11.5 (s, 1H, NH), 8.10-7.40(m, 16H, arom., H-6), 6.23 (d, 1H, H-11′), 5.41 (m, 1H, H-2′), 5.28 (m,1H, H-3′), 4.50-4.20 (m, 3H, H-4′, H-5′), 0.15 (s, 9H, Si(CH₃)₃).

In an analogous manner to the preparation of compound 36,1-(2′,3′,5′-tri-O-benzoyl-β-D-arabinofuranosyl)-5-[(2-trimethylsilyl)ethynyl]uracil42 was prepared from 29. ¹H NMR (DMSO-d₆) δ 11.3 (s, 1H, NH), 8.10-7.40(m, 16H, arom., H-6), 6.23 (d, 1H, H-1′), 5.40 (m, 1H, H-2′), 5.28 (m,1H, H-3′), 4.50-4.20 (m, 3H, H-4′, H-5′), 0.16 (s, 9H, Si(CH₃)₃).

In an analogous manner to the preparation of compound 36,1-[[1,3-bis(acetoxy)-2-propoxy]methyl]-5-[(2-trimethylsilyl)ethynyl]uracil50 was prepared from 47. ¹H NMR (CDCl₃) δ 8.44 (br, 1H, NH), 7.64 (s,1H, H-6), 5.27 (s, 2H, CH₂N), 4.21, 4.06 (2m, 5H, 2 CH₂, CH), 2.09 (s,6H, 2 CH₃), 0.23 (s, 9H, Si(CH₃)₃).

In an analogous manner to the preparation of compound 36,(±)-(1′β,3′α,4′β)-1-[3-acetoxy-4-(acetoxymethyl)cyclopentyl]-5-[(2-trimethylsilyl)ethynyl]uracil78 was prepared from 75. ¹H NMR (DMSO-d₆) δ 11.4 (bs, 1H, NH), 8.40 (s,1H, H-6), 5.1-4.5 (m, 2H, CHO, CHN), 4.30 (m, 2H, CH₂O), 2.02 (s, 6H, 2CH₃), 2.25-1.05 (m, 5H, 2 CH₂, CH), 0.16 (s, 9H, Si(CH₃)₃).

In an analogous manner to the preparation of compound 36,(2R,4R)-1-[2-(benzyloxymethyl)-1,3-dioxolan-4-yl]-5-(2-trimethylsilylethynyl)uracil97 was prepared from 94. ¹H NMR (CDCl₃) δ 8.30 (s, 1H, H-6), 8.20-7.40(m, 5H, arom.), 6.25 (m, 1H, H-2′), 5.20 (m, 1H, H-4′), 4.30-4.10 (m,4H, H-5′, H-6′), 0.15 (s, 9H, 3 CH₃).

In an analogous manner to the preparation of compound 36,(2S,4S)-1-[2-(benzyloxymethyl)-1,3-dioxolan-4-yl]-5-(2-trimethylsilylethynyl)uracil105 was prepared from 102. ¹H NMR (CDCl₃) δ 8.30 (s, 1H, H-6), 8.20-7.40(m, 5H, arom.), 6.25 (m, 1H, H-2′), 5.20 (m, 1H, H-4′), 4.30-4.10 (m,4H, H-5′, H-6′), 0.15 (s, 9H, 3 CH₃).

Example 9 β-D-2′-Deoxy-5-ethynyluridine (37)

To a mixture of 36 (1.333 g, 2.5 mmol) in THF (10 mL) was added TBAF (1M solution in THF, 2.5 mL, 2.5 mmol) and the mixture was stirred at roomtemperature for 30 minutes. After removal of the solvent, the residuewas dissolved in CH₂Cl₂, washed with brine and concentrated. The residuewas dissolved in NH₃-MeOH (2.0 M, 50 mL) and kept at room temperaturefor 20 h. After removal of the solvent by evaporation, the residue waspurified by flash chromatography on silica gel eluting with CH₂Cl₂/MeOH(9:1) to give the title compound 37 as a pale yellow solid (453 mg,72%). ¹H NMR (DMSO-d₆) δ 11.65 (s, 1H, NH), 8.30 (s, 1H, H-6), 6.10 (t,1H, H-1′), 5.26 (d, 1H, OH-3′), 5.15 (t, 1H, OH-5′), 4.23 (m, 1H, H-3′),4.12 (s, 1H, ethynyl), 3.79 (m, 1H, H-4′), 3.61 (m, 2H, H-5′), 2.13 (m,2H, H-2′).

In an analogous manner to the preparation of compound 37,β-L-2′-deoxy-5-ethynyluridine 39 was prepared from 38. ¹H NMR (DMSO-d₆)δ 11.65 (s, 1H, NH), 8.30 (s, 1H, H-6), 6.10 (t, 1H, H-1′), 5.26 (d,1H,)H-3′), 5.15 (t, 1H, OH-5′), 4.23 (m, 1H, H-3′), 4.12 (s, 1H,ethynyl), 3.79 (m, 1H, H-4′), 3.61 (m, 2H, H-5′), 2.13 (m, 2H, H-2′).

In an analogous manner to the preparation of compound 37,1-(β-L-arabinofuranosyl)-5-ethynyluracil 41 was prepared from 40. ¹H NMR(DMSO-d₆) δ 11.69 (s, 1H, NH), 8.37 (s, 1H, H-6), 6.02 (d, J=3.6 Hz, 1H,H-1′), 5.65, 5.54 (2d, 2H, 20H), 5.08 (t, 1H, OH-5′), 4.02-3.99 (m, 2H,H-2′, ethynyl), 3.95-3.93 (m, 1H, H-3′), 3.82-3.78 (m, 1H, H-4′),3.66-3.55 (m, 2H, H-5′).

In an analogous manner to the preparation of compound 37,1-(β-D-arabinofuranosyl)-5-ethynyluracil 43 was prepared from 42. ¹H NMR(DMSO-d₆) δ 11.7 (s, 1H, NH), 8.37 (s, 1H, H-6), 6.02 (d, 1H, H-1′),5.65, 5.54 (2d, 2H, 20H), 5.08 (t, 1H, OH-5′), 4.02-3.99 (m, 2H, H-2′,ethynyl), 3.95-3.93 (m, 1H, H-3′), 3.82-3.78 (m, 1H, H-4′), 3.66-3.55(m, 2H, H-5′).

In an analogous manner to the preparation of compound 37,1-[(1,3-dihydroxy-2-propoxy)methyl]-5-ethynyluracil 51 was prepared from50. ¹H NMR (DMSO-d₆) δ 11.63 (br, 1H, NH), 8.45 (s, 1H, H-6), 7.20, 6.60(2br, 2H, 20H), 5.18 (s, 2H, CH₂N), 4.66 (t, 2H, CH₂O), 4.12 (s, 1H,ethynyl), 3.54 (m, 1H, CHO), 3.40 (m, 2H, 2 CH₂O).

In an analogous manner to the preparation of compound 37,(±)-(1′β,3′α,4β)-1-[3-hydroxy-4-(hydroxymethyl)cyclopentyl]-5-ethynyluracil79 was prepared from 78. ¹H NMR (DMSO-d₆) δ 11.3 (bs, 1H, NH), 8.42 (s,1H, H-6), 5.00-4.55 (m, 3H, CHN, 20H), 4.10 (s, 1H, ethynyl), 4.00 (m,1H, CHOH), 3.42 (m, 2H, CH₂OH), 2.25-1.40 (m, 5H).

In an analogous manner to the preparation of compound 37,(2R,3R)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]-5-ethynyluracil 98 wasprepared from 97. ¹H NMR (DMSO-d₆) δ 11.38 (br, 1H, NH), 7.80 (s, 1H,H-6), 6.19 (m, 1H, H-2′), 5.61 (m, 1H, H-4′), 4.89 (br, 1H, OH),4.22-4.05 (m, 3H, H-5′, ethynyl), 3.13 (m, 1H, H-6′).

In an analogous manner to the preparation of compound 37,(2S,4S)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]-5-ethynyluracil 106 wasprepared from 105. ¹H NMR (DMSO-d₆) δ 11.38 (br, 1H, NH), 7.80 (s, 1H,H-6), 6.19 (m, 1H, H-2′), 5.61 (m, 1H, H-4′), 4.89 (br, 1H, OH),4.22-4.05 (m, 3H, H-5′, ethynyl), 3.13 (m, 1H, H-6′).

Example 10 1-[[1,3-Bis(benzyloxy)-2-propoxy]methyl]-5-iodouracil (45)

To a suspension of 2-O-(acetoxymethyl)-1,3-di-O-benzylglycerol (44;prepared according to Martin J. C., Dvorak C. A., Smee D. F., MatthewsT. R., Verheyden J. P. H. (1983)9-[(1,3-Dihydroxy-2-propoxy)methyl]quanine: a new potent and selectiveantiherpes agent, J. Med. Chem. 26, 759-761) (2.038 g, 5.9 mmol) and5-iodouracil (2.106 g, 8.85 mmol) in anhydrous CH₂Cl₂ (25 mL) at roomtemperature was added N,O-bis-(trimethylsilyl)acetamide (BSA, 5.46 mL,22.13 mmol), and the mixture was stirred at room temperature undernitrogen for 2 h. The resulting clear solution was cooled to 0° C., andSnCl₄ (1 M solution in CH₂Cl₂, 5.9 mL, 5.9 mmol) was added. The mixturewas stirred at room temperature overnight, and then poured into amixture of saturated aqueous NaHCO₃ and CHCl₃. The aqueous layer wasextracted with CHCl₃, and the combined organic phase was evaporated. Theresidue was purified by flash chromatography on silica gel eluting withCH₂Cl₂/MeOH (97:3) to give the title compound 45 as a yellow oil (2.935g, 95%). ¹H NMR (CDCl₃) δ 8.59 (s, 1H, NH), 7.82 (s, 1H, H-6), 7.36-7.26(m, 10H, arom.), 5.28 (s, 2H, CH₂N), 4.50 (s, 4H, 2 CH₂), 3.99 (m, 1H,CHO), 3.52 (m, 4H, 2 CH₂O).

Example 11 1-[[1,3-Dihydroxy-2-propoxy]methyl]-5-iodouracil (46)

To a solution of 45 (1.90 g, 3.64 mmol) in anhydrous CH₂Cl₂ (60 mL) at−78° C. was added BCl₃ (1 M solution in CH₂Cl₂, 30 mL, 30 mmol)dropwise. The mixture was stirred at −78° C. under nitrogen for 2 h,then at −60° C. for another 4 h. MeOH (10 mL) was added to quench thereaction, followed by 14% NH₄OH solution to adjust the mixture to pH 7.After being stirred at room temperature for 1 h, the mixture wasconcentrated in vacuo. The residue was purified by flash chromatographyon silica gel eluting with CH₂Cl₂/MeOH (9:1 to 4:1) to give the titlecompound 46 as a pale yellow solid (810 mg, 65%). ¹H NMR (DMSO-d₆) δ11.6(s, 1H, NH), 8.19 (s, 1H, H-6), 7.20, 6.60 (2br, 2H, 20H), 5.15 (s, 2H,CH₂N), 4.60 (m, 2H, CH₂O), 3.53 (m, 1H, CHO), 3.41 (m, 2H, 2 CH₂O).

Example 12 1-[[1,3-Bis(acetoxy)-2-propoxy]methyl]-5-iodouracil (47)

To a solution of 46 (400 mg, 1.17 mmol), DMAP (10 mg), Et₃N (1.0 mL) inanhydrous CH₂Cl₂ (7 mL) at 0° C. was added Ac₂O (0.5 mL) dropwise, andthe solution was stirred at room temperature under nitrogen overnight.After removal of the solvent by evaporation, the residue was purified byflash chromatography on silica gel eluting with CH₂Cl₂/MeOH (97:3) togive the title compound 47 as a yellow oil (499 mg, quantitative). ¹HNMR (CDCl₃) δ 8.66 (br, 1H, NH), 7.78 (s, 1H, H-6), 5.26 (s, 2H, CH₂N),4.23, 4.05 (2m, 5H, 2 CH₂, CHO), 2.07 (s, 6H, 2 CH₃).

In an analogous manner to the preparation of compound 47,(±)-(1′β,3′α,4′β)-1-[3-acetoxy-4-(acetoxymethyl)cyclopentyl]-5-iodouracil75 was prepared from 72. ¹H NMR (DMSO-d₆) δ 11.2 (bs, 1H, NH), 8.13 (s,1H, H-6), 5.00 (m, 1H, CHO), 4.90 (m, 1H, CHN), 4.18, 4.05 (2m, 2H,CH₂O), 2.02 (s, 6H, 2 CH₃), 2.45-1.85 (m, 4H, 2 CH₂), 1.59 (m, 1H, CH).

Example 13 β-D-2′-Deoxy-3′,5′-di-O-p-toluoyl-5-iodo-4′-thiouridine (55)

A suspension of 5-iodorouracil (1.071 g, 4.5 mmol) and ammonium sulfate(5 mg) in HMDS (30 mL) was heated at reflux under nitrogen atmospherefor 2 h. The excess of HMDS was evaporated in vacuo. To the residue wasadded a solution of1-O-acetyl-2-deoxy-3,5-di-O-p-toluoyl-4-thio-α/β-D-ribofuranoside (54;prepared according to Secrist III J. A., Tiwari K. N., Riodan J. M.,Montgomery J. A. (1991) Synthesis and biological activity of21′-deoxy-4′-thio pyrimidine nucleosides, J. Med. Chem. 34, 2361-2366)(1.284 g, 3 mmol) in anhydrous CH₃CN (20 mL). The resulting mixture wascooled to 0° C., and TMSOTf (907 mg, 4 mmol) was added. The reactionmixture was stirred at 0° C. for 20 min, then at room temperature for 2h. The mixture was diluted with CH₂Cl₂, washed with saturated NaHCO₃,dried over Na₂SO₄, filtered and concentrated to dryness. The residue waspurified by flash chromatography on silica gel eluting with CH₂Cl₂/MeOH(99:1), and recrystallized from CH₂Cl₂/hexane to give the title compound55 as a solid (563 mg, 31%). ¹H NMR (CDCl₃) δ 7.96-7.25 (m, 9H, arom.,H-6), 6.68 (dd, 1H, H-1′), 5.75 (m, 1H, H-3′), 4.68 (m, 2H, H-5′), 4.05(m, 1H, H-4′), 2.75, 2.41 (2m, 2H, H-2′), 2.42 (s, 6H, 2 CH₃).

In an analogous manner to the preparation of compound 55,β-D-2′-deoxy-3′,5′-di-O-p-toluoyl-4′-thio-5-E-[(2-trimethylsilyl)ethynyl]uridine58 was prepared from 54 and 5-[(2-trimethylsilyl)ethynyl]uracil(prepared according to Imamura K. & Yamamoto Y. (1997) Synthesis and invitro evaluation of 5-closo- and 5-nido-orthocarboranyluridines as boroncarriers, Bull. Chem. Soc. Jpn. 70, 3103-3110). ¹H NMR (CDCl₃) δ 8.36(s, 11H, H-6), 7.94-7.26 (m, 9H, arom.), 6.70 (m, 1H, H-1′), 5.73 (m,1H, H-3′), 4.70 (m, 2H, H-5′), 4.05 (m, 1H, H-4′), 2.76, 2.40 (2m, 2H,H-2′), 2.42 (s, 6H, 2 CH₃), 0.14 (s, 9H, Si(CH₃)₃).

In an analogous manner to the preparation of compound 55,β-D-5-E-(2-chlorovinyl)-2′-deoxy-3′,5′-di-O-p-toluoyl-4′-thiouridine 60was prepared from 54 and 5-E-(2-chlorovinyl)uracil (prepared accordingto Jones A. S., Verhelst G., Walker R. T. (1979) The synthesis of thepotent anti-herpes virus agent, E-5-(2-bromovinyl)-2′-deoxyuridine andrelated compounds, Tetrahedron Lett. 45, 4415-4418). ¹H NMR (DMSO-d₆) δ11.6 (s, 1H, NH), 8.10 (s, 1H, H-6), 7.95-7.85 (m, 4H, arom.), 7.40-7.20(m, 4H, arom.), 7.14 (d, J=13 Hz, 1H, vinyl), 6.64 (d, J=13 Hz, 1H,vinyl), 6.40 (t, 1H, H-1′), 5.82 (m, 1H, H-3′), 4.70-4.50 (m, 2H, H-5′),3.95 (m, 1H, H-4′), 2.80-2.50 (m, 2H, H-2′), 2.40 (s, 6H, 2 CH₃).

In an analogous manner to the preparation of compound 55,5-iodo-1-(4-thio-2,3,5-tri-O-benzyl-β-D-arabinofuranosyl)uracil 63 wasprepared from1-O-acetyl-4-thio-2,3,5-tri-O-benzyl-α/β-D-arabinofuranoside (62;prepared according to Secrist III J. A., Tiwari K. N., Shortnacy-FowlerA. T., Messini L., Riodan J. M., Montgomery J. A., (1998) Synthesis andbiological activity of certain 4′-thio-D-arabinofuranosylpurinenucleosides, J. Med. Chem. 41, 3865-3871). ¹H NMR (CDCl₃) δ 8.10 (br,1H, NH), 7.80 (s, 1H, H-6), 7.40-7.15 (m, 15H, arom.), 6.30 (d, 1H,H-1′), 4.70-4.40 (m, 6H, 3 CH₂), 4.20 (m, 2H, H-2′, H-3′), 3.65 (m, 2H,H-5′), 3.40 (m, 1H, H-4′).

In an analogous manner to the preparation of compound 55,1-(4-thio-2,3,5-tri-O-benzyl-β-D-arabinofuranosyl)-5-[(2-trimethylsilyl)ethynyl]uracil66 was prepared from 62. ¹H NMR (CDCl₃) δ 8.30 (s, 1H, H-6), 7.42-7.15(m, 15H, arom.), 6.29 (d, 1H, H-1′), 4.70-4.40 (m, 6H, 3 CH₂), 4.18 (m,2H, 2′, H-3′), 3.65 (m, 2H, H-5′), 3.40 (m, 1H, H-4′), 0.14 (s, 9H,Si(CH₃)₃).

In an analogous manner to the preparation of compound 55,5-E-(2-chlorovinyl)-1-(4-thio-2,3,5-tri-O-benzyl-β-D-arabinofuranosyl)uracil68 was prepared from 62. ¹H NMR (CDCl₃) δ 8.2 (br, 1H, NH), 7.75 (s, 1H,H-6), 7.40-7.15 (m, 16H, arom., vinyl), 6.28 (d, 1H, H-1′), 6.00 (d, 1H,J=13 Hz, vinyl), 4.70-4.40 (m, 6H, 3 CH₂), 4.20 (m, 2H, H-2′, H-3′),3.65 (m, 2H, H-5′), 3.40 (m, 1H, H-4′).

Example 14 β-D-2′-Deoxy-4′-thio-5-vinyluridine (57)

To a mixture of 56 (220 mg, 0.43 mmol) in MeOH (5 mL) was added NaOMe(1.0 M solution, 0.87 mL, 0.87 mmol), and the mixture was stirred atroom temperature for 1 h. The solvent was evaporated, and the residuewas purified by flash chromatography on silica gel eluting withCH₂Cl₂/MeOH (9:1) to give the title compound 57 as a white solid (78 mg,67%). ¹H NMR (DMSO-d₆) δ 11.3 (s, 1H, NH), 8.21 (s, 1H, H-6), 6.4 (dd,1H, CH═CH₂), 6.28 (t, 1H, H-1′), 6.00, 5.15 (2d, 2H, CH═CH₂), 5.23 (m,2H, 20H), 4.35 (m, 1H, H-3′), 3.64 (m, 2H, H-5′), 3.30 (m, 1H, H-4′),2.35-2.15 (m, 2H, H-2′).

In an analogous manner to the preparation of compound 57,β-D-5-E-(2-chlorovinyl)-2′-deoxy-4′-thiouridine 61 was prepared from 60.¹H NMR (DMSO-d₆) δ 11.6 (s, 1H, NH), 8.18 (s, 1H, H-6), 7.23 (d, J=13Hz, 1H, vinyl), 6.70 (d, J=13 Hz, 1H, vinyl), 6.25 (t, 1H, H-1′), 5.27(d, 1H, OH-3′), 5.21 (t, 1H, 1H, OH-5′), 4.35 (m, 1H, H-3′), 3.70-3.15(m, 3H, H-5′, H-4′), 2.30-2.10 (m, 2H, H-2′).

In an analogous manner to the preparation of compound 57,β-D-2′-deoxy-5-ethyluridine 124 was prepared from 123. ¹H NMR (DMSO-d₆)δ 11.3 (s, 1H, NH), 7.69 (s, 1H, H-6), 6.18 (t, 1H, J=6.8 Hz, H-1′),5.24 (d, 1H, J=4.0 Hz, OH-3′), 5.06 (t, 1H, J=4.8 Hz, OH-5′), 4.25 (m,1H, H-3′), 3.77 (m, H-4′), 3.57 (m, 2H, H-5′), 2.21 (q, 2H, CH₂), 2.09(m, 2H, H-2′), 1.03 (t, 3H, CH₃).

In an analogous manner to the preparation of compound 57,β-D-5-butyl-2′-deoxyuridine 126 was prepared from 125. ¹H NMR (DMSO-d₆)δ 11.24 (bs, 1H, NH), 7.68 (s, 1H, H-6), 6.16 (t, 1H, J=6.8 Hz, H-1′),5.23, 5.04 (2bs, 2H, 20H), 4.23 (m, 1H, H-3′), 3.75 (m, H-4′), 3.55 (m,2H, H-5′), 2.17 (q, 2H, CH₂), 2.07 (m, 2H, H-2′), 1.38, 1.26 (2m, 4H, 2CH₂), 0.86 (t, 3H, CH₃).

Example 15 β-D-2′-Deoxy-5-ethynyl-4′-thiouridine (59)

To a mixture of 58 (440 mg, 0.76 mmol) in THF (5 mL) was added TBAF (1 Msolution in THF, 0.76 mL, 0.76 mmol) and the mixture was stirred at roomtemperature for 30 min. After removal of the solvent, the residue wasdissolved in CH₂Cl₂, washed with brine dried (Na₂SO₄), and concentrated.The residue was dissolved in MeOH (5 mL) and NaOMe (1.0 M solution inMeOH, 1.53 mL) was added. The solution was stirred at room temperaturefor 1 h, and then the solvent was evaporated. The residue was purifiedby flash chromatography on silica gel eluting with CH₂Cl₂/MeOH (9:1) togive the title compound 59 as a pale yellow solid (98 mg, 48%). ¹H NMR(DMSO-d₆) δ 11.4 (br, 1H, NH), 8.5 (s, 1H, H-6), 6.25 (t, 1H, H-1′),5.20 (m, 2H, 20H), 4.32 (m, 1H, H-3′), 4.20 (s, 1H, ethynyl), 3.62 (m,2H, H-5′), 3.32 (m, 1H, H-4′), 2.30-2.10 (m, 2H, H-2′).

Example 16 1-(4-Thio-β-D-arabinofuranosyl)-5-vinyluracil (65)

To a solution of 64 (664 mg, 1.5 mmol) in anhydrous CH₂Cl₂ (10 mL) at−78° C. was added slowly BBr₃ (1 M solution in CH₂Cl₂, 7.5 mL), and thereaction mixture was stirred at −78° C. under nitrogen atmosphere for 4h. The reaction was quenche by addition of MeOH (5 mL), and neutralizedby pyridine. After removal of the solvent by evaporation, the residuewas purified by flash chromatography on silica gel eluting withCH₂Cl₂/MeOH (4:1) to give the title compound 65 (303 mg, 77%). ¹H NMR(DMSO-d₆) δ 11.2 (s, 1H, NH), 8.20 (s, 1H, H-6), 6.4 (dd, 1H, CH═CH₂),6.27 (d, 1H, H-1′), 6.00, 5.15 (2d, 2H, CH═CH₂), 5.70 (d, 1H, OH-2′),5.40 (d, 1H, OH-3′), 5.27 (t, 1H, OH-5′), 4.00-3.95 (m, 2H, H-2′, H-3′),3.75-3.65 (m, 2H, H-5′), 3.20 (m, 1H, H-4′).

In an analogous manner to the preparation of compound 65,5-E-(2-chlorovinyl)-1-(4-thio-β-D-arabinofuranosyl)uracil 69 wasprepared from 68. ¹H NMR (DMSO-d₆) δ 11.5 (s, 11H, NH), 8.20 (s, 11H,H-6), 7.23 (d, J=13 Hz, 1H, vinyl), 6.70 (d, J=13 Hz, 1H, vinyl), 6.26(d, 1H, H-1′), 5.68 (d, 1H, OH-2′), 5.40 (d, 1H, OH-3′), 5.25 (t, 1H,OH-5′), 4.02-3.95 (m, 2H, H-2′, H-3′), 3.75-3.60 (m, 2H, H-5′), 3.21 (m,1H, H-4′).

Example 17 5-Ethynyl-1-(4-thio-β-D-arabinofuranosyl)uracil (67)

To a mixture of 66 (940 mg, 1.5 mmol) in THF (10 mL) was added TBAF (1 Msolution in THF, 1.5 mL, 1.5 mmol), and the mixture was stirred at roomtemperature for 30 min. After removal of the solvent, the residue wasdissolved in CH₂Cl₂ (50 mL), washed with water, dried over Na₂SO₄, andevaporated to dryness in vacuo. Then the residue was dissolved inanhydrous CH₂Cl₂ (10 mL), and cooled to −78° C. BBr₃ (1 M solution inCH₂Cl₂, 7.5 mL) was added slowly, and the reaction mixture was stirredat −78° C. under nitrogen atmosphere for 4 h. The reaction was quenchedby addition of MeOH (5 mL), and neutralized by pyridine. After removalof the solvent by evaporation, the residue was purified by flashchromatography on silica gel eluting with CH₂Cl₂/MeOH (4:1) to give thetitle compound 67 (311 mg, 73%). ¹H NMR (DMSO-d₆) δ 11.3 (br, 1H, NH),8.42 (s, 1H, H-6), 6.25 (t, 1H, H-1′), 5.68 (br, 1H, OH-2′), 5.42 (d,1H, OH-3′), 5.25 (t, 1H, OH-5′), 4.10 (s, 1H, ethynyl), 4.01-3.94 (m,2H, H-2′, H-3′), 3.72-3.60 (m, 2H, H-5′), 3.24 (m, 1H, H-4′).

Example 18 β-D-5-(5-Chlorothien-2-yl)-2′-deoxyuridine (84)

A solution of 81 (618 mg, 1.1 mmol) and NCS (142 mg, 1.05 mmol) inanhydrous pyridine (10 mL) was heated at 70° C. for 4 h. After removalof the solvent, the residue was co-evaporated with toluene, and then asolution of NH₃-MeOH (2 M solution, 40 mL) was added. The solution wasstirred in a stoppered flask at room temperature overnight. Afterremoval of the solvent, the residue was purified by flash chromatographyon silica gel eluting with CH₂Cl₂/MeOH (9:1) to give, afterrecrystallization from acetone/CHCl₃, the title compound 84 as abrownish-yellow crystal (208 mg, 55%). ¹H NMR (DMSO-d₆) δ 11.8 (s, 1H,NH), 8.67 (s, 1H, H-6), 7.24 (d, 1H, J=4.4 Hz, thienyl), 7.06 (d, 1H,J=4.4 Hz, thienyl), 6.19 (t, 1H, J=6.0 Hz, H-1′), 5.37 (t, 1H, J=4.8 Hz,OH-5′), 5.29 (d, 1H, J=4.4 Hz, OH-3′), 4.31 (m, 1H, H-3′), 3.84 (m, 1H,H-4′), 3.71-3.65 (m, 2H, H-5′), 2.25-2.18 (m, 2H, H-2′).

In an analogous manner to the preparation of compound 84,β-L-5-(5-chlorothien-2-yl)-2′-deoxyuridine 91 was prepared from 88. ¹HNMR (DMSO-d₆) δ 11.8 (s, 1H, NH), 8.67 (s, 1H, H-6), 7.24 (d, 1H, J=4.4Hz, thienyl), 7.06 (d, 1H, J=4.4 Hz, thienyl), 6.19 (t, 1H, J=6.0 Hz,H-1′), 5.37 (t, 1H, J=4.8 Hz, OH-5′), 5.29 (d, 1H, J=4.4 Hz, OH-3′),4.31 (m, 1H, H-3′), 3.84 (m, 1H, H-4′), 3.71-3.65 (m, 2H, H-5′),2.25-2.17 (m, 2H, H-2′).

Example 19 β-D-5-(5-Bromothien-2-yl)-2′-deoxyuridine (85)

To a mixture of 81 (113 mg, 0.2 mmol) and CHCl₃ (10 mL) at roomtemperature was added Br₂—CCl₄ (32 mg Br₂ in 2 mL CCl₄) dropwise over aperiod of 15 min, and then the solution washed twice with water. Theorganic phase was evaporated and co-evaporated with toluene, and then asolution of NH₃-MeOH (2 M solution, 30 mL) was added. The solution wasstirred in a stoppered flask at room temperature overnight. Afterremoval of the solvent, the residue was purified by flash chromatographyon silica gel, eluting with CH₂Cl₂/MeOH (9:1) to give, afterrecrystallization from acetone/CHCl₃, the title compound 85 as a lightyellow solid (49 mg, 63%). ¹H NMR (DMSO-d₆) δ 11.8 (s, 1H, NH), 8.67 (s,1H, H-6), 7.21 (d, 1H, J=4.0 Hz, thienyl), 7.16 (d, 1H, J=4.0 Hz,thienyl), 6.19 (t, 1H, J=6.0 Hz, H-1′), 5.37 (t, 1H, J=4.8 Hz, OH-5′),5.29 (d, 1H, J=4.4 Hz, OH-3′), 4.30 (m, 1H, H-3′), 3.84 (m, 1H, H-4′),3.71-3.65 (m, 2H, H-5′), 2.25-2.18 (m, 2H, H-2′).

In an analogous manner to the preparation of compound 85,β-L-5-(5-bromothien-2-yl)-2′-deoxyuridine 92 was prepared from 88. ¹HNMR (DMSO-d₆) δ 11.8 (s, 1H, NH), 8.67 (s, 1H, H-6), 7.21 (d, 1H, J=4.0Hz, thienyl), 7.16 (d, 1H, J=4.0 Hz, thienyl), 6.19 (t, 1H, J=6.4 Hz,H-1′), 5.36 (t, 1H, J=4.4 Hz, OH-5′), 5.28 (d, 1H, J=4.4 Hz, OH-3′),4.30 (m, 1H, H-3′), 3.83 (m, 1H, H-4′), 3.70-3.65 (m, 2H, H-5′),2.25-2.17 (m, 2H, H-2′).

In an analogous manner to the preparation of compound 85,(2R,4R)-5-(5-bromo-2-furanyl)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]uracil113 was prepared from 110. ¹H NMR (DMSO-d₆) δ 11.5 (s, 1H, NH), 8.26 (s,1H, H-6), 6.82, 6.60 (2d, 2H, furanyl), 6.19 (m, 1H, H-2′), 5.62 (m, 1H,H-4′), 4.90 (br, 1H, OH), 4.20-4.05 (m, 2H, H-5′), 3.12 (m, 1H, H-6′).

In an analogous manner to the preparation of compound 85,(2R,4R)-5-(5-bromothien-2-yl)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]uracil115 was prepared from 111. ¹H NMR (DMSO-d₆) δ 11.8 (br, 1H, NH), 8.65(s, 1H, H-6), 7.20 (d, 1H, thienyl), 7.16 (d, 1H, thienyl), 6.19 (m, 1H,H-2′), 5.61 (m, 1H, H-4′), 4.89 (br, 1H, OH), 4.22-4.05 (m, 2H, H-5′),3.13 (m, 1H, H-6′).

In an analogous manner to the preparation of compound 85,(2S,4S)-5-(5-bromo-2-furanyl)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]uracil119 was prepared from 116. ¹H NMR (DMSO-d₆) δ 11.5 (s, 1H, NH), 8.26 (s,1H, H-6), 6.82, 6.60 (2d, 2H, furanyl), 6.19 (m, 1H, H-2′), 5.62 (m, 1H,H-4′), 4.90 (br, 1H, OH), 4.20-4.05 (m, 2H, H-5′), 3.12 (m, 1H, H-6′).

In an analogous manner to the preparation of compound 85,(2S,4S)-5-(5-bromothien-2-yl)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]uracil121 was prepared from 117. ¹H NMR (DMSO-d₆) δ 11.8 (br, 1H, NH), 8.65(s, 1H, H-6), 7.20 (d, 1H, thienyl), 7.16 (d, 1H, thienyl), 6.19 (m, 1H,H-2′), 5.61 (m, 1H, H-4′), 4.89 (br, 1H, OH), 4.22-4.05 (m, 2H, H-5′),3.13 (m, 1H, H-6′).

Example 20 β-D-2′-Deoxy-5-(5-iodothien-2-yl)uridine (86)

To a mixture of 81 (225 mg, 0.4 mmol) in anhydrous MeCN (10 mL) wasadded 12, followed by ceric ammonium nitrate (61 mg, 0.113 mmol). Themixture was stirred at room temperature for 3 h, and then the solventwas evaporated. The residue was taken into EtOAc (30 mL), 5% sodiumhydrosulfite (5 mL), and brine (10 mL). The aqueous layer was extractedwith EtOAc, and the combined organic phase was concentrated to dryness.Due was co-evaporated with toluene and EtOH, and then dissolved in 2 MN₃-MeOH solution (30 mL). The solution was stirred in a stoppered flaskat room temperature overnight. After removal of the solvent, the residuewas purified by flash chromatography on silica gel eluting withCH₂Cl₂/MeOH (9:1) to give, after recrystallization fromMeOH/acetone/CHCl₃, the title compound 86 as a brown solid (96 mg, 55%).¹H NMR (DMSO-d₆) δ 11.8 (s, 1H, NH), 8.64 (s, 1H, H-6), 7.25 (d, 1H,J=4.0 Hz, thienyl), 7.11 (d, 1H, J=4.0 Hz, thienyl), 6.19 (t, 1H, J=6.4Hz, H-1′), 5.36 (t, 1H, J=4.8 Hz, OH-5′), 5.29 (d, 1H, J=4.4 Hz, OH-3′),4.30 (m, 1H, H-3′), 3.84 (m, 1H, H-4′), 3.70-3.64 (m, 2H, H-5′),2.26-2.18 (m, 2H, H-2′).

In an analogous manner to the preparation of compound 86,β-L-2′-deoxy-5-(5-iodothien-2-yl)uridine 93 was prepared from 88. ¹H NMR(DMSO-d₆) δ 11.8 (s, 1H, NH), 8.64 (s, 1H, H-6), 7.25 (d, 1H, J=4.0 Hz,thienyl), 7.11 (d, 1H, J=4.0 Hz, thienyl), 6.19 (t, 1H, J=6.4 Hz, H-1′),5.36 (t, 1H, J=4.8 Hz, OH-5′), 5.29 (d, 1H, J=4.4 Hz, OH-3′), 4.30 (m,1H, H-3′), 3.84 (m, 1H, H-4′), 3.70-3.64 (m, 2H, H-5′), 2.25-2.17 (m,2H, H-2′).

Example 21(2R,4R)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]-5-(3-hydroxypropenyl)uracil(101)

To a solution of 100 (447 mg, 1.5 mmol) in toluene (10 mL) at −78° C.was added DIBALH (1.0 M solution in hexane, 1.8 mL) in a period of 10min. After stirring at −78° C. for 30 min, the reaction was quenched byaddition of MeOH (2 mL), and the solution was allowed to warm to roomtemperature. Saturated sodium potassium tartrate solution was added andthe mixture was filtered through a short pad of celite. The filtrate wasconcentrated in vacuo and the residue was purified by flashchromatography on silica gel, eluting with CH₂Cl₂/MeOH (9:1) to give thetitle compound 101 as a white solid (226 mg, 56%). ¹H NMR (DMSO-d₆) δ11.5 (s, 1H, NH), 7.62 (s, 1H, H-6), 6.25 (d, 1H, H-2′), 6.09 (d, 1H,vinyl), 5.73 (m, 1H, vinyl), 5.20 (t, 1H, H-4′), 4.90 (m, 2H, 20H),4.29, 4.08 (m, 4H, H-5′, CH₂), 3.64(dd, 2H, H-6′).

In an analogous manner to the preparation of compound 100,(2S,4S)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]-5-(3-hydroxypropenyl)uracil109 was prepared from 108. ¹H NMR (DMSO-d₆) δ 11.5 (s, 1H, NH), 7.62 (s,1H, H-6), 6.25 (d, 1H, H-2′), 6.09 (d, 1H, vinyl), 5.73 (m, 1H, vinyl),5.20 (t, 1H, H-4′), 4.90 (m, 2H, 20H), 4.29, 4.08 (m, 4H, H-5′, CH₂),3.64(dd, 2H, H-6′).

Example 22 β-D-2′-Deoxy-35′-di-0-p-toluoyl-5-ethyluridine (123)

A suspension of 5-ethyluracil (420 mg, 3 mmol) and ammonium sulfate (15mg) in HMDS (20 mL) was heated at reflux under nitrogen atmosphere for 2h. The excess of HMDS was evaporated in vacuo. To the residue was addedCHCl₃ (10 mL), followed by2-deoxy-3,5-di-O-p-toluoyl-α-D-erythro-pentofuranosyl chloride (122; 778mg, 2 mmol; prepared according to Hoffer M, Chem. Ber. 1960, 93, 2771)portionwise, with stirring at room temperature. The resulting solutionwas stirred at room temperature overnight. The solvent was concentratedto half the original volume, and EtOH was added. The rest of CHCl₃ wasevaporated, the precipitate was filtered, washed with EtOH, and dried invacuo to give the title compound 123 as a white powder (890 mg, 90%). ¹HNMR (CDCl₃) δ 8.08 (s, 1H, NH), 7.97-7.92 (m, 4H, arom.), 7.30-7.27 (m,4H, arom.), 7.22 (s, 1H, H-6), 6.46 (dd, 1H, H-1′), 5.65 (d, 1H, H-3′),4.82-4.62 (m, 2H, H-5′), 4.54 (m, 1H, H-4′), 2.70, 2.34 (2m, 2H, H-2′),2.45 (d, 6H, 2 CH₃), 2.07 (q, 2H, CH₂), 0.89 (t, 3H, CH₃).

In an analogous manner to the preparation of compound 100,β-D-5-butyl-2′-deoxy-3′,5′-di-O-p-toluoyl-uridine 125 was prepared from122. ¹H NMR (CDCl₃) δ 8.15 (s, 1H, NH), 7.97-7.92 (m, 4H, arom.),7.30-7.27 (m, 4H, arom.), 7.23 (s, 1H, H-6), 6.46 (dd, 1H, H-1′), 5.65(d, 1H, H-3′), 4.81-4.61 (m, 2H, H-5′), 4.53 (m, 1H, H-4′), 2.70, 2.34(2m, 2H, H-2′), 2.44 (d, 6H, 2 CH₃), 2.02 (q, 2H, CH₂), 1.27-1.09 (m,4H, 2 CH₂), 0.79 (t, 3H, CH₃).

Example 23 β-D-2′-Deoxy-5-(hydroxymethyl)uridine (136)

To a solution of 2′-deoxyuridine (135; 10 g, 43.8 mmol) in 0.5 N KOH(100 mL) was added paraformaldehyde (12.5 g), and the mixture was heatedat 60° C. for 1 day. Additional 0.5 N KOH (150 mL) was added, and themixture was stirred at 60° C. for 6 days. The mixture was neutralizedwith Dowex-50WX-8-100, and filtered. The resin was rinsed with water,and the combined filtrate was concentrated in vacuo. The oily residuewas purified by flash chromatography on silica gel eluting withCHCl₃/MeOH (4:1) to give the title compound 136 as a yellow sticky foam(4.30 g, 38%). ¹H NMR (DMSO-d₆) δ 11.4 (br, 1H, NH), 7.78 (s, 1H, H-6),6.16 (m, 1H, H-1′), 4.24 (m, 1H, H-3′), 4.12 (s, 2H, CH₂), 3.76 (m,H-4′), 3.57 (m, 2H, H-5′), 2.06 (m, 2H, H-2′).

Example 24

Competition Assay of Thymidine Phosphorylation

This assay is an indirect measure of the relative affinity the enzymehas for each nucleoside and is used here to investigate the recognitionof a nucleoside analogue by the EBV-TK (Gustafson E. A., Chillemi A. C.,Sage D. R., Fingeroth J. (1998) The EBV-TK does not phosphorylate GCV orACV and demonstrates a narrow substrate specificity compared to theHSV-1 TK. Antimicrob. Agents Chemother. 42, 2923-2931; Gustafson E. A.,Schinazi R. F., Fingeroth J. (2000) HHV-8 open reading frame 21 is athymidine and thymidylate kinase of narrow substrate specificity thatefficiently phosphorylates zidovudine but not ganciclovir. J. Virol. 74,684-692). EBV-TK was incubated in an optimized buffer with 15 μM 3H-dTas substrate in a final volume of 100 μL. The enzyme source was eitherlysate from 143B EBV-TK-expressing cells or purified GST EBV-TK from E.coli. The amount of enzyme was adjusted so that the reaction proceededlinearly for over 60 min. A 10-fold excess (150 μM) of the nucleosideanalog being tested was included in the experiment. The reaction washalted at 30 min. by spotting 50 μL of the reaction onto positivelycharged DE-81 discs (Whatman). After washing four times with 5 mMammonium formate and once with 95% ethanol, the discs were dried andcounted in a scintillation counter. The CPM of the positive controlreaction containing no nucleoside analog was taken as 100% activity.Effective competition with the labeled substrate ³H-dT resulted in alower CPM value.

Example 25

Cytotoxicity Assay

This assay measures the ability of a candidate nucleoside tospecifically eliminate EBV-infected cells in the continuous presence ofdrug. 143B T-cells lack TK-1. 293 cells contain human TK-1. Therefore,these assays comparatively assess the effects of human TK expression ontoxicity. Stock solutions (20-40 mM) of the compounds were prepared insterile DMSO and then diluted to the desired concentration in growthmedium. The cells (143B T-, 293 EBV-TK or 293 Neo control cells) werecultured in the presence of drug for 5 days, and the media was replacedwith fresh media containing drug on day three. Cells were then washedone time with PBS, fixed with 4% formaldehyde/1% methanol and stainedwith 1% crystal violet in 20% ethanol for visualization of the cellmonolayer. To score the toxicity, “+” means that the cells did notsurvive, “−” indicates that the cells grew at the highest concentrationof drug tested, and “+/−” means that the cells were still alive, but notreplicating or replicating very slowly. Cultures with this designationoften have media that is still “pink,” indicating a slowdown of cellularmetabolism. Identical samples are assessed in triplicate.

Example 26

Colony Formation Assay

This assay is designed to identify nucleosides whose toxicity is due toincorporation into cellular DNA forming a lethal lesion following asingle exposure to the drug, as described for GCV (Rubsam L. Z.,Davidson B. L., Shewach D. S. (1998) Superior cytotoxicity with GCVcompared with ACV and 1-beta-D-arabinofuranosylthymine inHSV-TK-expressing cells: a paradigm for cell killing, Cancer Res. 58,3873-3882). Stock solutions (20-40 mM) of the compounds were prepared insterile DMSO and then diluted to the desired concentration in growthmedium. Exponentially growing cells were incubated with the nucleosidein question for 24 h, plated in 24-well plates at 50 viable cells perwell and incubated for 7-9 days in the absence of drug. Cells were fixedand stained as above and colonies were counted. A nucleoside can cause adecrease in the number of colonies, which is an indirect indication ofthe number of cells able to survive and to replicate after the initialexposure to drug.

Example 27

Anti-HIV and Cytotoxicity Assay

Anti-HIV-1 activity of the compounds was determined in human peripheralblood mononuclear (PBM) cells as described previously (Schinazi R. F.,McMillan A., Cannon D., Mathis R., Lloyd R. M. Jr., Peck A., SommadossiJ.-P., St. Clair M., Wilson J., Furman P. A., Painter G., Choi W.-B.,Liotta D. C. Antimicrob. Agents Chemother. 1992, 36, 2423; Schinazi, R.F., Sommadossi J.-P., Saalmann V., Cannon D., Xie M.-Y., Hart G., SmithG., Hahn E. Antimicrob. Agents Chemother. 1990, 34, 1061). Stocksolutions (20-40 mM) of the compounds were prepared in sterile DMSO andthen diluted to the desired concentration in growth medium. Cells wereinfected with the prototype HIV-1LAI at a multiplicity of infection of0.01. Virus obtained from the cell supernatant was quantified on day 6after infection by a reverse transcriptase assay using (rA)n•(dT)12-18as template-primer. The DMSO present in the diluted solution (<0.1%) hadno effect on the virus yield. AZT was included as positive control. Thetoxicity of some compounds was assessed in Vero, human PBM, and CEMcells, as described previously (Schinazi R. F., Sommadossi J.-P.,Saalmann V., Cannon D. L., Xie M.-Y., Hart G. C., Smith G. A., Hahn E.F. Antimicrob Agents Chemother. 1990, 34, 1061-1067). Cycloheximide wasincluded as positive cytotoxic control, and untreated cells exposed tosolvent were included as negative controls. The antiviral EC₅0, EC₉₀,and cytotoxicity IC₅₀ were obtained from the concentration-responsecurve using the median effective method described previously (ChouT.-C.; Talalay P. Adv. Enzyme Regul. 1984, 22, 27-55; Belen'kii M. S.,Schinazi R. F. Antiviral Res. 1994, 25, 1-11).

Example 28

Production of Cells Expressing EBV-TK (or KHSV-TK)

The Epstein-Barr virus (EBV) thymidine kinase (TK) gene from the viralstrain B-958 was cloned into the vector pCMV as described (Gustafson EA, Chillemi A C, Sage D R, Fingeroth J D. The Epstein-Barr virusthymidine kinase does not phosphorylate ganciclovir or acyclovir anddemonstrates a narrow substrate specificity compared to the herpessimplex virus type 1 thymidine kinase. Antimicrob Agents Chemother 1998;42(11), 2923-31). The vector was then transfected using Lipofectaminereagent into 2 human cell lines. The cells were selected and expressionof the TK gene (RNA and protein) was determined. The cells were used tocreate two assay systems to assess the ability of EBV-TK to sensitizecells to candidate nucleoside analogs. Cells that expressed KHSV-TK weresimilarly prepared for assay.

Example 29

Production of 143B T-human Osteosarcoma Cells that Express EBV-TK (orKHSV-TK)

143B T-osteosarcoma cells were obtained from ATCC. These cells contain amutated human TK1 gene and do not synthesize human TK1 RNA/protein. Theydo synthesize human TK 2 and thymidylate synthase. They are maintainedin media containing 5-bromodeoxyuridine when not in use, to preventreversion to a TK⁺ phenotype. The cells cannot utilize the thymidinesalvage pathway unless a protein with effective thymidine kinaseactivity is introduced. Cells transfected with EBV-TK were selected withHAT (hypoxanthine, aminopterin, thymidine). The aminopterin component,an antifolate blocks reactions involving tetrahydrofolate in the normalde novo synthesis of purine nucleoside monophosphates and thymidinemonophosphate. This produces reliance on the salvage pathways forsurvival.

EBV-TK transfectants grew as clones. Both multiple individual clones andbulk populations (a mixture of clones) were utilized in differentexperiments. Cells that were transfected with an empty control vectorthat did not express TK did not grow at all in HAT.

All cells that survived selection expressed EBV-TK. This was documentedby 2 methods (1) detection of EBV-TK RNA (Northern blot hybridization orRNA dot blot hybridization using the EBV-TK containing genomic fragmentBXLF1 as probe) and/or (2) detection of EBV-TK protein (immunoblot usinghuman heteroantisera from patients with nasopharyngeal carcinoma knownto have high titer antibody to EBV-TK or more recently with amonospecific rabbit heteroantisera to purified EBV-TK). EBV-TK-bearingcells were utilized shortly after selection and frozen. Transfectantswere repeatedly documented to be human TK1 negative by RNA blothybridization. Additional control cell lines were also established. 143BT-cells were similarly transfected with HSV-1 TK, KHSV (KSHV) TK andhuman TK-1. Preparation of the respective plasmids used for transfectionwas as described in Gustafson E A, Chillemi A C, Sage D R, and FingerothJ D. The Epstein-Barr virus thymidine kinase does not phosphorylateganciclovir or acyclovir and demonstrates a narrow substrate specificitycompared to the herpes simplex virus type 1 thymidine kinase. AntimicrobAgents Chemother 1998; 42(11), 2923-31 and Gustafson E A, Schinazi R F,and Fingeroth J D. Human herpesvirus 8 open reading frame 21 is athymidine and thymidylate kinase of narrow substrate specificity thatefficiently phosphorylates zidovudine but not ganciclovir. J Virol 2000;74(2), 684-92). Expression was documented by RNA blot hybridization.

To eliminate the need for HAT selection in some experimentsEBV-TK-bearing 143B TK-cells (and controls i.e. 143B T-cells alone or143B T-cells expressing respectively HSV1-TK, KHSV-TK, human TK1 wereremoved from HAT selection and retransfected with the plasmid pSV2 neowhich confers resistance to Geneticin (G418). The cells were thenselected with G418. Clonal expression of EBV-TK was documented byNorthern blot hybridization and/or by immunoblot. Cells were reanalyzedas indicated prior to relevant experiments. These cells were used toscreen candidate nucleoside analogs. They provide an effective screen asthey clearly discriminate the activities provided by the respective TKsin an in vitro assay. Compounds that are toxic to EBV-TK-expressingcells but not toxic to 143B T cells or to 143B human TK1-expressingcells (in the presence or absence of HAT selection) are demonstrated tohave EBV-TK specific cytotoxic activity. The activity of candidatecompounds is further compared with HSV1-TK and KHSV-TK-expressing 143BTK-cells, providing information that may have additional therapeuticapplications.

Example 30

Production of 293 Human Embryonic Kidney Cells that Express EBV-TK (orKHSV-TK)

293 cells were obtained from ATCC. These cells are human embryonickidney cells immortalized with sheared adenovirus DNA and express theadenovirus proteins E1A and E1B. 293 cells express human TK1 and TK2.For production of 293 EBV-TK cells, 293 cells were co-transfected withpCMV EBV-TK and pSV2 neo or with pCMV control and pSV2 neo at a 20:1ratio (typically 2-10 μg of pCMV EBV-TK and 0.1-0.5 μg pSV2neo). Thetransferred cells were selected with G418 (1 mg/ml). Clones weredocumented to express EBV-TK as described above.

293 KHSV-TK cells were prepared similarly using the vector pCMV KHSV-TKtogether with pSV2 neo (co-transfection) or the single vectorpcDNA3-KHSV-TK (which contains an endogenous selectable neo marker).Clones were documented to express KHSV-TK by RNA blot hybridization.Positive clones were also pooled to produce bulk populations.

293 EBV-TK neo (clones and bulk) were used to screen candidatenucleoside analogs in the 2 independent assays described in Examples 25and 26.

Example 31

Cytotoxicity Assays (143B T- and 293 Transfectants)

Two cell systems are employed to determine the toxicity of eachnucleoside. The information obtained depends upon the system used. In143B T-cells, cytotoxic drugs that are dependent upon phosphorylation bya TK are easily identified. 143B T-cells expressing either EBV-TK orhTK1 permit assessment of whether either enzyme independently may causea drug to become cytotoxic. Thus, drugs that can be activated by hTK1,which would cause non-specific cytotoxicity, are readily identified. Onedrawback is that these cells are grown in HAT media that “forces”nucleosides through the TK salvage pathway (EBV-TK or hTK1). A falsepositive may arise if a nucleoside out-competes dT for phosphorylationsuch that sufficient amounts of dT are not available for cellularreplication. This toxicity may not be present or may be reduced in acell that has the synthetic as well as salvage pathways available fordTTP production. For this reason, the 293 system was developed. Thesecells have endogenous hTK1 and EBV-TK is maintained by selection inG418. Thus, the normal hTK1 salvage pathway is operational, andnucleosides can be expected to flux through the cells normally. Drugsare incubated with 293 cells transformed with a neomycin (Neo)expressing plasmid (Neo control) and with cells transfected with thesame plasmid also expressing EBV-TK. A nucleoside selectively activatedby EBV-TK should cause toxicity on 293 EBV-TK and not on 293 Neo controlcells. In both the 143B and 293 cell systems, toxicity is evaluated bycell survival. The cells are cultured in the presence of drug for 5days, and the media replaced with fresh media containing drug on day 3.Cells are then washed once with PBS, fixed with 4% formaldehyde/1%methanol and stained with 1% crystal violet in 20% ethanol forvisualization of the cell monolayer. To score the toxicity, “+” meansthat the cells did not survive, “−” indicates that the cells grew fineat the highest concentration of drug tested, and “+/−” means that thecells were still alive, but not replicating or replicating very slowly(media remains “pink,” indicating a slowdown of cellular metabolism).Direct quantitation was performed by counting of cells in the presenceof trypan blue stain to exclude dead cells (blinded technician). Allassays were performed in triplicate. Although other methods areavailable, trypan blue exclusion has provided a superior measurement ofcell death when compared with assays such as MTT that effectivelydocument growth inhibition, but not death.

Example 32

Colony Formation Assay (293 Transfectants)

This assay is designed to identify nucleosides whose toxicity is due toincorporation into cellular DNA forming a lethal lesion as described forGCV (Rubsam L Z, Davidson B L, and Shewach D S. “Superior cytotoxicitywith ganciclovir compared with acyclovir and1-β-D-arabinofuranosylthymine in herpes simplex virus-thymidinekinase-expressing cells: a novel paradigm for cell killing” Cancer Res1998; 58(17):3873-82). Such toxicity is not dependent upon constantexposure to drug, but only exposure to drug during log phase growth.Nucleosides that act via a different mechanism, such as chaintermination or inhibition of an enzyme in a metabolic pathway, are oftenonly cytostatic, and the cells may return to normal growth after drugremoval. Unlike the general cytotoxicity screen, such nucleosides do notdemonstrate increased cytotoxicity in this assay. Exponentially growingcells are incubated with the candidate nucleoside for 24 hours, platedin 24-well plates at 50 viable cells/well and incubated for 7-9 days inthe absence of drug. Cells are fixed and stained as above and coloniesare counted. A nucleoside that works effectively causes a decrease inthe number of colonies, which is a direct indication of the number ofcells able to survive and replicate after the initial exposure to drug.

This invention has been described with reference to its preferredembodiments. Variations and modifications of the invention, will beobvious to those skilled in the art from the foregoing detaileddescription of the invention. It is intended that all of thesevariations and modifications be included within the scope of thisinvention.

1. A pharmaceutical composition comprising an effective amount to treata cell carrying the Epstein-Barr virus of KHSV of a compound selectedfrom among(2S,4S)-5-(2-furanyl)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]uracil,(2S,4S)-5-(5-bromo-2-furanyl)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]uracil,(2S,4S)-5-(2-thienyl)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]uracil,(2S,4S)-5-(5-bromthien-2-yl)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]uracil,and(2S,4S)-5-(3-hydroxypropenyl)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]uracil.2. The pharmaceutical composition of claim 1 wherein the compound is(2S,4S)-5-(2-furanyl)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]uracil. 3.The pharmaceutical composition of claim 1 wherein the compound is(2S,4S)-5-(5-bromo-2-furanyl)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]uracil.4. The pharmaceutical composition of claim 1 wherein the compound is(2S,4S)-5-(2-thienyl)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]uracil. 5.The pharmaceutical composition of claim 1 wherein the compound is(2S,4S)-5-(5-bromthien-2-yl)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]uracil.6. The pharmaceutical composition of claim 1 wherein the compound is(2S,4S)-5-(3-hydroxypropenyl)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]uracil.